Journal of the Egyptian Society of Parasitology, Vol.45, No.3, December 2015

J. Egypt. Soc. Parasitol. (JESP), 45(3), 2015: 593-602

ANTIPARASITIC ACTIVITY OF SILVER AND COPPER OXIDE NANOPARTICLES AGAINST HISTOLYTICA AND CRYPTOSPORIDIUM PARVUM CYSTS BY ABDEL HALIM A. SAAD1, MOHAMED I. SOLIMAN1, AHMED M. AZZAM2* and AMR B. MOSTAFA1 Department of Zoology1, Faculty of Science, Ain-Shams University, Cairo 11566 and Department of Environmental Researches2, Theodor Bilharz Research Institute, >Imbaba P.O. Box 30, Giza 12411, Egypt (*Correspondence:

Abstract Nanoparticles (NPs) have received more attention as antiparasitic agents. In the present study, silver and copper nanoparticles were synthesized and characterized using scanning electron microscopy (SEM), transmission electron microscope (TEM) and X-ray fluorescence (XRF). The antiparasitic activity of Ag and CuO nanoparticles were tested against two of the most environmentally spread parasites in Egypt (Entamoeba histolytica and Cryptosporidium parvum). The average sizes of synthesized Ag NPs and CuO NPs were 9 & 29 nm respective- ly and a significant reduction for cysts viability (p > 0.05) was observed for CuO NPs against E. histolytica cysts and Ag NPs against C. parvum oocysts. Moreover, LC50-3h of CuO NPs for E. histolytica and C. parvum were 0.13 and 0.72 mg/l, while Ag NPs recorded 0.34 and 0.54 mg/l respectively. Accordingly, these NPs could be suggested as a new nanoform agent for safe and effective treatment of E. histolytica and C. parvum parasites. Keywords: Antiparasitic, silver, copper, nanoparticles, Entamoeba histolytica, Cryptospo- ridium parvum.

Introduction children below 5 years of age G. duodenalis Amaebiasis, a major health problem in de- and C. parvum were the only parasites de- veloping countries, is the second most com- tected; 7.0 and 5.0 %, respectively (El- mon cause of death due to parasitic infec- Mohammady et al, 2012). tion. Amaebiasis is usually transmitted by The main motive is the expectation that the ingestion of Entamoeba cysts through nanoparticles will be able to be used in the oral–fecal route (Shukla et al, 2015). Among treatment of various diseases in the future the , Entamoeba, Giardia and Cryp- (Angeli et al, 2008; Debbage, 2009). It was tosporidia are the major causes of water- determined that through their unique proper- borne diseases (Marshall et al, 1997; Sharma ties and large surface areas, metal oxide na- et al, 2003). The prevalence of E. histolytica noparticles possess effective among outpatients in the Dakahlia Gover- activities (Elechiguerra et al, 2005). Particu- norate in Egypt was 9.7% (El Shazly et al, larly, owing to their great chemical reactivi- 2006). Besides, Cryptosporidium parvum ty, nanoparticles are capable of producing is a leading cause of diarrhea in reactive oxygen species (ROS), which have Egypt and its prevalence among diarrheic the ability to kill infectious agents. individuals whether outpatient or inpatient The use of metal oxide nanoparticles ex- clinics was up to 49% (Youssef et al, 2008; hibiting the antimicrobial activity offers the Mousa et al, 2010; Helmy et al, 2013). possibility of an efficient removal of patho- Moreover, the commonest protozoan infec- gens from wastewater (Elechiguerra et al, tion in immunosuppressed patients was C. 2005). The NPs may not have the pro- parvum which reached 60.2% (Abdel-Ha- nounced antimicrobial activity when com- feez et al, 2012). In Cairo and the Nile Delta pared to the bulk formulations of the metal between 2005 and 2007 applied ELISA oxide or solutions of metal salts. But, the technique for the detection of bacterial, viral stability and slow release of metal ions from and parasitic pathogens in 2,112 diarrheic nanoparticles are main characteristics which

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give them the advantage in use (Heinlaan et times with deionized water under nitrogen al, 2008). stream. A dried powder of the nanosize sil- The antimicrobial efficiency of NPs de- ver was obtained by freeze-drying (Guzmán pends on the particle size (Adams et al, et al, 2009). 2006). The smallest sized of NPs showed the Preparation of copper oxide NPs: Aqueous strongest effect (Lu et al, 2013). Some re- solution (250ml) of copper acetate (0.02mol) searches indicate that silver nanoparticles, (Merck Company) is prepared in round bot- gold, chitosan, and oxidized metals have tom flask. 1 ml glacial acetic acid is added growth inhibitory or cytotoxic effect on var- to above aqueous solution and heated to ious parasites, including Giardia, Leishma- 100ºC with constant stirring. About 0.4gm nia, Malaria, Toxoplasma and insect larva NaOH (El-Gomhouria Chemical Co., Egypt) (Elmi et al, 2013). Also, Ag NPs showed was added to above heated solution till the significant anti-leishmanial effects by inhib- pH reached 6-7. The large amount of black iting the promastigotes proliferation and me- precipitate was formed immediately, centri- ta-bolic activity (Allahverdiyev et al, 2011). fuged, washed 3-4 times with deionized wa- Therefore, nanoparticles are recommended ter and the precipitate was air dried in for 24 for destroying parasites (cytotoxic and inhib- h (Lanje et al, 2010). itory effect), because they act as more effec- Characterization of nanoparticles: Nano- tive and less harmful and also benefi- particles prepared were characterized with cial for the prevention and control the help of multiple techniques. The surface of the parasites (Elmi et al, 2013). morphology of nanoparticles was character- The aim of the present study was to deter- ized by a scanning electron microscopy mine the efficacy of synthesized Ag and (SEM; JEOL JSM-5600). The particles size CuO nanoparticles in treatment of Entamoe- of the resulted nanoparticles was analyzed ba histolytica and Cryptosporidium parvum. by using transmission electron microscope

Subjects, Materials and Methods (TEM) (EM 208S Philips, Netherlands) Preparation of silver nanoparticles: For the connected to a high resolution imaging sys- preparation of silver nanoparticles two stabi- tem. Samples for TEM studies were pre- lizing agents, sodium dodecyl sulphate pared by placing drops of nanoparticles so- (SDS) (Sigma-Aldrich Company) and sodi- lutions on carbon-coated TEM copper grids. um citrate (Merck Company) were used. For X-ray fluorescence (XRF) that was per- the synthesis of silver nanoparticles, silver formed to detect the main chemical ele- nitrate solution (1.0mM) and 8% (w/w) SDS mental analysis of the minerals that are pre- were used as a metal salt precursor and a sent in synthesized nanoparticles. XRF mea- stabilizing agent. Hydrazine hydrate (Sigma- surements were carried out using the JSX- Aldrich Company) solution with a concen- 3222 element analyzer. tration 2.0mM and citrate of sodium solution Collection of cysts: E. histolytica cysts and (1.0mM) were used as a reducing agents. C. parvum oocysts were isolated from posi- Citrate of sodium was also used as stabiliz- tive samples of farmers and their animals’ ing agent at room temperature. The transpar- feces from three different governorates Cai- ent colorless solution was converted to the ro, Giza and Qaluobia. All samples were characteristic pale yellow and pale red color, used directly after arrival. A highly purified when citrate of sodium was used as stabiliz- cysts suspension was achieved by combining ing agent. The occurrence of color was indi- the sucrose flotation method with a simpli- cated the formation of silver nanoparticles. fied sucrose gradient method (Sheffield and The silver nanoparticles were purified by Bjorvatn, 1977). The feces were broken up centrifugation. To remove excess silver ions, in normal saline (0.9%) and filtered through the silver colloids were washed at least three a 300µm filter. A total of 3ml of feces sus-

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pension was layered on top of 3ml of 0.85M ences (SPSS 18.0). The differences between sucrose (Sigma-Aldrich Company) and cen- treated and untreated samples were tested trifuged at 2,000 for 10 min at 4°C. The for significance using a t- test (P ˂ 0.05) was cysts were aspirated with a Pasteur pipette at considered statistically significant. sucrose-water interface and washed 3 times Results with normal saline. The washed cysts were Silver nanoparticles synthesis and cha- carefully added to the top of a discontinuous racterization: The scanning electron micro- density gradient consisting of two 3ml layers scope images showed that Ag NPs were of 0.85M and 0.4M sucrose. After centrifu- round-shaped with a smooth surface mor- gation, the cysts concentrated at the 0.85- phology and tended to form aggregates (Fig. 0.4M sucrose interface were collected and 1A). The particle size analysis of the nano- washed again. The purified cysts were sus- particles was performed by transmission pended in normal saline and stored at 4°C electron microscopy, where TEM images of for a maximum of 3 days prior to use. the aqueous dispersion showed average par- Anti-parasitic activity: The nanoparticles ticle size 9nm (Fig. 1B). The elemental ana- were prepared in four concentrations (0.5, lysis of Ag NPs was performed using XRF 1.0, 2.0 & 4.0mg/l). A total of 2ml of each analysis of powder showed that 98.4% of solution was placed in test tubes, to which metal composition of Ag NPs powder was washed cysts were added and gently mixed. Ag (Fig. 1E). The tubes were then incubated at 37ºC for Copper oxide nanoparticles synthesis 10, 30, 60 & 180 min for each concentration. At the end of each incubation period, the and characterization: SEM image showed upper phase was carefully removed. Un- spherical nanoparticles morphology (Fig. treated cysts were used as a control group in 1C). TEM photos showed that the CuO NPs each experiment. A total of 2ml of 0.1% eo- are spherical and its average diameter is 29 sin stain was then added to the remaining nm (Fig. 1D). XRF recorded that 98.5% of settled cysts, and gently mixed. Eosin 0.1% metal composition of CuO NPs powder is stain was used to detect the cysts’ viability Cu (Fig. 1F). Antiparasitic activity: The ac- for 15min. The cysts were then smeared on a tivity of two nanoparticles showed a signi- glass slide, covered with a cover glass and ficant difference between untreated groups examined under a light microscope. Dead and treated ones. Time as an effective pa- cysts percent was determined as 100 cysts/ rameter influenced mortality rate of cysts; slide. Cysts without absorbed dye were rec- increasing the duration of exposure also in- orded as potentially viable; otherwise rec- creased the mortality rate. orded as dead one. The tests were repeated Entamoeba histolytica cysts recorded high in triplicate (Yarahmadi et al, 2014). mortality rates after 180 min of exposure SEM of treated C. parvum oocysts: Con- time ranged from 73% to 100 % for CuO trol and treated C. parvum oocysts by NPs NPs (Fig. 3A) and from 51% to 100 % for were fixed by using fresh 2% glutaraldehyde Ag NPs (Fig. 3B). Difference between for an hour, rinsed in sodium cacodylate groups treated with NPs and control was buffer and post fixed for an hour at 4°C with significant (P˂0.05) at concentrations 2.0 1% osmium tetraoxide in sodium cacodylate and 4.0 mg/l for Ag NPs (Fig. 3F), and at buffer solution. Suspensions were progressi- 1.0, 2.0 and 4.0 mg/l for CuO NPs (Fig. 3E). vely dehydrated. The preparation were coat- No significant difference was observed be- ed with gold salt and examined with a SEM; tween the treated and untreated groups at 0.5 JEOL JSM-5600 (Brasseur et al, 1998). mg/l of exposure time for both nanoparticles Statistical analysis: Data were analyzed and 1.0 mg/l for Ag NPs. The mortality rates using statistical package for the social sci- of cysts treated with Ag and CuO nanoparti- cles after 180 min of exposure time by a

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Fig. 1: A) SEM of Ag NPs, B) TEM of Ag NPs, C) SEM of CuO NPs, D) TEM of CuO NPs, E) XRF of Ag NPs, F) XRF of CuO NPs.

Fig. 2: SEM images of control and treated oocysts of C. parvum by nanoparticles.

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concentration of 0.5mg/l were 51% and 63 The antibacterial effects of Ag NPs were %, by the side of 1.0 mg/l was 85 and 100% recorded (Zheng et al, 2008). Silver nano- respectively, while on 2.0mg/l and 4.0mg/l particles, gold and oxidized metals have mortality rate was 100% for both NPs (Fig. growth inhibitory or cytotoxic effect on var- 3E, F). But, the mortality rate of the control ious parasites, including the Giardia, Leish- group at 180 min was 8%. The present re- mania, Plasmodium, Toxoplasma and insect sults showed that mortality rate increased larva (Elmi et al, 2013). Also, the Ag NPs gradually with the increase in Ag NPs and showed significant anti-leishmanial effects CuO NPs concentrations (Fig. 3A, B). Addi- by inhibition of promastigotes proliferation tionally, LC50-3h values of NPs for E. histo- and metabolic activity (Allahverdiyev et al, lytica cysts were 0.13 and 0.34 mg/l with 2011). CuO and Ag, respectively, which indicated Time as an effective parameter influenced that E. histolytica cysts more sensitive for the mortality rate of parasites cysts; increas- CuO than Ag NPs. Mortality rates of C. par- ing the exposure time, improved the mortali- vum oocysts after 180 min of contact time ty rate. Also, in the present study, E. histo- mortality ranged from 39% to 100% for lytica and C. parvum oocysts showed gradu- CuO NPs (Fig. 3C) and from 47% to 100% al increase in mortality rate with the increase for Ag NPs (Fig. 3D). Difference between in Ag NPs & CuO NPs concentrations. The groups treated with Ag NPs and CuO NPs LC50-3h of nanoparticles for E. histolytica and control was significant (P˂0.05) at con- cysts were 0.13 and 0.34 mg/l with CuO and centrations 2.0 and 4.0mg/l for Ag NPs (Fig. Ag, respectively indicating that E. histolyti- 3F), but CuO NPs recorded significant re- ca cysts sensitive for CuO was more than duction at 4.0mg/l (Fig. 3E). The mortality Ag NPs. LC50-3h values of nanoparticles for rate of C. parvum oocysts treated with Ag C. parvum oocysts were 0.54 and 0.72 mg/l NPs and CuO NPs after 180 min of exposure with Ag and CuO, respectively indicating time at a concentration of 0.5mg/l were 47% that C. parvum sensitive for Ag was more and 39%, by 1.0mg/l was 73% & 59%, by than CuO NPs. The present results agreed the side of 2.0mg/l was 100 and 81% respec- with Allahverdiyev et al. (2011) who found tively, while on 4.0mg/l mortality rate was that Ag NPs recorded the highest suppress- 100% for both NPs (Fig. 3C, D). The mor- ive effect on parasites at a concentration of tality rate of control group at 180 min was 2.0mg/L, and at same concentration, the me- 9%. The study results showed that the mor- tabolic activity of Leishmania tropica pro- tality rate increased gradually with the in- mastigotes decreased nearly twofold, in con- crease in Ag NPs & CuO NPs concentra- trast to the control group (P˂0.05). Smith tions (Fig. 3E, F). Also, LC50-3h values of (2013) in mice studied the silver nanoparti- Ag NPs for C. parvum were 0.54 mg/l, cles effects on C. parvum using a murine while CuO NPs was 0.72 mg/l, which indi- model and dielectrophoresis. They found cated that C. parvum more sensitive for Ag that ionic silver released from the silver NPs than CuO NPs. SEM images of C. parvum has a modest but measureable effect on C. oocysts treated with Ag & CuO nanoparti- parvum infectivity. However, Liao et al. cles showed changes in the oocyst wall’ (2012) revealed that C. parvum oocysts structure compared to control ones (Fig. 2). monitoring method into a powerful scale due

Discussion to Ag NPs treatment performed within very short time and its nontoxic agent property. In the present study, Ag and CuO nanopar- So, accepted to be a good waterborne proto- ticles showed anti-parasitic activity. Where- zoan parasite determination method, rapidly as, E. histolytica and C. parvum cysts rec- diagnose and prevent the infectious out- orded high mortality rates after 180 min of break. Su et al. (2014) used in vitro excysta- exposure time for Ag NPs and CuO NPs.

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tion and fluorogenic assays to examine dis- plain why copper nanoparticles exert toxic infection capabilities of silver nanoparticles effects on eukaryotic cells. They reviewed on C. parvum oocysts and validated their that nanoparticles can diffuse into the cell results by in vivo animal infectivity assays. directly through the pores present in cell They found that almost two orders of magni- membrane due to their small size, or they get tude reduction in oocyst shedding among entry through ion channels and transporter mice fed on silver nanoparticle treated oo- proteins present on the plasma membrane. cysts relative to untreated ones. Thus, CuO Some nanoparticles may enter into cells via NPs and Ag NPs could be suggested as a endocytosis. They found that the nanoparti- new nanoform agent as safe and effective cles which are entered into the cell can di- treatment of E. histolytica and C. parvum rectly interact with oxidative organelles such besides, NPs were biologically non-toxic. as mitochondria. Later, redox active proteins However, Blinova et al. (2010) compared stimulate ROS production in cells, and ions 2+ the CuO NPs toxicity with CuSO4 in natural (Cu ) produced by nanoparticles can induce river waters samples collected in six differ- ROS by various chemical reactions. ROS ent sites. They found that LC50-48h ranged can induce DNA strand breaks, and affect from 92.7 to >200mg/l for CuO NPs and gene expression. Schrand et al. (2010) hy- 0.24 to 0.92mg/l to CuSO4. The lower tox- pothesized that copper nanoparticles act as icity in river water samples as compared to effective antibacterial agent against wide the test media was attributed to the presence range of bacterial species due to interactions of organic matter that can strongly complex with -SH groups leading to protein denatura- to Cu and reduce the Cu ions bioavailability. tion. Copper nanoparticles exert effect on In the present study, SEM images of C. cell membrane due to affinity towards amin- parvum showed that oocysts became inac- es and carboxyl groups on organisms’ cell tive after treatment by NPs, where changes surface as Bacillus subtilis (Beve- in the structure of its wall were observed ridge and Murray 1980; Ren et al, 2009). after treated with NPs compared to control, Once inside the cell, the nanoparticles might which indicated that these oocysts loss its bind with DNA molecules and disturb the viability. Choi and Hu (2008) consider the helical structure by cross-linking within & reason for this is the interaction of NPs with between nucleic acid strands (Stohs and the surface of parasites. It may be posited Bagchi, 1995). Copper ions inside bacterial that NPs impair the structure of lipophos- cells also disrupt biochemical processes phoglycan and glycoprotein molecules that (Kim et al, 2000). are found on the surface of parasites and The particle size analysis of prepared na- which are responsible for the infection. They noparticles by TEM showed average particle also proposed that these molecules may be size 9 & 29 nm for Ag NPs & CuO NPs re- more seriously affected from ROS generated spectively, while SEM images showed that form NPs and this may lead to inhibition of Ag NPs were round-shaped with a smooth parasite infection. Also, Choi and Hu (2008) surface morphology and tended to form ag- reported that owing to their great chemical gregates and CuO NPs were spherical shape. reactivity, nanoparticles are capable of ROS, Allahverdiyev et al. (2011) revealed that Ag which have the ability to kill infectious NPs of size ranged 10-40 nm demonstrated agents. The main reason for using Ag NPs in significant anti-leishmanial effects by inhib- this study was their capacity to produce iting proliferation and metabolic activity of ROS. Moreover, Chang et al. (2012) dis- promastigotes. Nevertheless, antimicrobial cussed three different mechanisms based on activity of Cu NPs with an average size of oxidative stress, coordination effects, and 20 nm reported significant inhibitory activity non-homeostasis effects that potentially ex- against Escherichia coli followed by Klebsi-

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Fig. 3: Mortality rates at different exposure time of A) Treated E. histolytica cysts by CuO NPs, B) E. histolytica cysts by Ag NPs, C) Treated C. parvum oocysts by CuO NPs, D) Treated C. parvum oocysts by Ag NPs, Mortal- ity rates after 180 min of exposure time for both parasites treated by E) CuO NPs F) Ag NPs.

Fig. 4: Images of treated parasites by NPs and stained by 0.1% eosin A) Viable E. histolytica cyst B) Non-viable E. histolytica cyst C) Viable C. parvum oocyst D) Non-viable C. parvum oocyst.

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ella pneumoniae, Pseudomonas aerugino- tion of baboons by Entamoeba histolytica elicits sa, Propionibacterium acnes and Salmonella anti- gal-lectin heavy subunit IgA and IgG anti- typhi (Shende et al, 2015). Generally, the bodies with shared epitope specificity to that of antimicrobial efficiency of NPs depends on humans. J. Egypt. Soc. Parasitol. 43, 3:723-35. the particle size (Adams et al, 2006), with Abdel-Hafeez, EH, Ahmad, AK, Ali, BA, Mo- slam, FA, 2012: Opportunistic parasites among the activities of Ag NPs were size-depen- immunosuppressed children in Minia District, dent; the smallest one showed the strongest Egypt. Korean J. Parasitol. 50:57-62. effect (Lu et al, 2013). Adams, LK, Lyon, DY, Alvarez, PJ, 2006: Co- Generally speaking, E. histolytica affects mparative eco-toxicity of nanoscale TiO2, SiO2, anyone, and commonest in people who live and ZnO water suspensions. Water Res. 40: in tropical areas (CDC, 2010). Besides, in 3527-32. Egypt E. histolytica was reported by many Allahverdiyev, AM, Abamor, ES, Bagirova, authors particularly in immunocompromized M, Ustundag, CB, Kaya, C, et al, 2011: Anti- patients (El-Beshbishi et al, 2005; Abd-Alla leishmanial effect of silver nanoparticles and et al, 2013; El Nadi et al, 2015; Foda and their enhanced antiparasitic activity under ultra- Singh, 2015; Banisch et al, 2015; El-Shazly violet light. Int. J. Nanomed. 6:2705-14. Angeli, E, Buzio, R, Firpo, G, Magrassi, R, et al, 2015). On the other hand, the zoonotic Mussi, V, et al 2008: Nanotechnology applica- was reported in nearly all tions in medicine. Tumori 94, 2:206-15. the Egyptian Governorates from man partic- Azab, ME, Khalil, HM, Khalifa, AS, Makh- ularly in the immunocompromized patients, louf, SA, Habib, SM, 1985: Cryptosporidiosis animals and even water sources (Azab et al, as a possible cause of diarrhea in children. Eg- 1985; Soliman, 1992; Youssef et al, 1994; ypt. J. Paediat. 2: 547-353. El Shazly et al, 2007; El-Sherbini and Mo- Baiomy, AM, Mohamed, KA, Ghannam, M hammad, 2007; Massoud et al, 2008; Rayan A, Shahat. SA, Al-Saadawy, AS, 2010: Oppor- et al, 2009; Baiomy et al, 2010; Khalifa et tunistic parasitic among immunocom- al, 2014). promised Egyptian patients. J. Egypt. Soc. Para- Conclusion sitol. 40, 3: 797-808. Banisch, DM, El-Badry, A, Klinnert, JV, Ig- Undoubtedly, the gastrointestinal protozo- natius, R, El-Dib, N, 2015: Simultaneous detec- an parasites particularly, Entamoeba histo- tion of Entamoeba histolytica/dispar, Giardia lytica and Cryptosporidium parvum are duodenalis and cryptosporidia by immunochro- among the commonest causes of diarrheal matographic assay in stool samples from patients illness particularly among children world- living in the Greater Cairo Region, Egypt. World wide. Their prevalence in children with diar- J. Microbiol. Biotechnol. 31, 8:1251-8. rhea is higher than in those without them. Beveridge, TJ, Murray, RGE, 1980: Sites of The outcome results showed the capabil- metal deposition in the cell wall of bacillus sub- ity of CuO and Ag nanoparticles to inacti- tilis. J. Bacteriol. 141:876-7. vate E. histolytica and C. parvum cysts. The Blinova, I, Ivask, A, Heinlaan, M, Mortimer, CuO NPs and Ag NPs may represent an al- M, Kahru, A, 2010: Ecotoxicity of nanoparti- cles of CuO and ZnO in natural water. Environ. ternative drugs and water treatment for E. Pollut. 158, 1:41-7. histolytica and C. parvum cysts. The treat- Brasseur, P, Uguen, C, Moreno-Sabater, A, ment based on CuO NPs and Ag NPs have a Favennec, L, Ballet, JJ, 1998: Viability of Cry- very important role in overcoming amoebia- ptosporidium parvum oocysts in natural waters. sis and cryptosporidiosis. Extensive studies Folia Parasitol. 45, 2:113-6. on this issue are ongoing and while be pub- CDC, 2010: Parasites-amebiasis (known as E. lished in due time elsewhere. histolytica infection) Atlanta, GA 30333. References Chang, YN, Zhang, M, Xia, L, Zhang, J, Xi- ng, G, 2012: The toxic effects and mechanisms Abd-Alla, MD, Wolf, RF, White, GL, Kosan- of CuO and ZnO nanoparticles. Materials 5: ke, SD, Carey, DW, et al, 2013: Natural infec- 2850-71,

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