Antiprotozoal effects of metal nanoparticles against Ichthyophthirius multifiliis

SUMMARY Ichthyophthirius multifiliis is a widespread, ciliated protozoan ectoparasite of fish. In the present study, we investigated the effects of metal nanoparticles on the reproduction and infectivity of free-living stages of I. multifiliis. We determined that ~50% of theronts could be killed within 30 min of exposure to either 20 ng mL−1 gold, 10 ng mL−1 silver or 5 ng mL−1 zinc oxide nanoparticles. Silver and zinc oxide nanoparticles at concentration of 10 and 5 ng mL−1 killed 100 and 97% of theronts, respectively and inhibited reproduction of tomonts after 2 h exposure. Gold nanoparticles at 20 ng mL killed 80 and 78% of tomonts and theronts 2 h post exposure, respectively. In vivo exposure studies using rainbow trout (Oncoryhnchus mykiss) demonstrated that theronts, which survived zinc oxide nanoparticles exposure, showed reduced infectivity compared with control theronts. No mortalities were recorded in the fish groups cohabited with theronts exposed to either nanoparticles compared with 100% mortality in the control group. On the basis of the results obtained from this study, metal nanoparticles particularly silver nanoparticles hold the best promise for the development of effective antiprotozoal agents useful in the management of ichthyophthiriosis in aquaculture.

Ichthyophthirius multifiliis is a ciliated protozoan parasite with a worldwide distribution. It causes 'white spot disease' of freshwater fish, and is responsible for severe epizootics in aquaria, hatcheries, and ponds. The parasite has low host and tissue specificity, infecting body surfaces including gills, skin, eyes, and fins. Although the ciliate was well recognized in the Middle Ages, and possibly had its origin in cyprinid fishes imported from Asia, it was formally first described in carp and other freshwater fishes in central Europe (Hoffman, 1967). The parasite has worldwide distribution, which is primary correlated to human mediated introductions of alien host species (non-naive) into new environments (Hoffman, 1970), and has been reported in feral fishes in tropical and temperate regions (Nigrelli et al. 1976). The broad spatial distribution of the parasite is due in part, to the ability of the encysted tomonts to survive temperatures ranging from 2 to 27°C. Factors leading to epizootic include thermal triggers, the temperature tolerance of the host fish, and degree of resistance of the host (Nigrelli et al. 1976). Despite the widespread and costly impacts of the parasite, few successful management strategies have been developed to control I. multifiliis infections (Shinn et al. 2012). After theronts penetrate into fish skin and gills, control of the disease becomes difficult. Inhibiting the parasite while it is still a tomont, before it replicates, is crucial to stopping its spread (Tucker & Robinson, 1990;Schäperclaus, 1991;Fu et al. 2014). Chemicals and drugs currently used to control I. multifiliis have potential environmental and host toxicities, particularly the most efficacious, malachite green (Rintamäki-Kinnunen & Valtonen, 1997). This compound is now prohibited for use in food fish due to its carcinogenic and teratogenic properties, which leaves few other chemicals that are effective (Shinn et al. 2012). Thus, there is a crucial need to investigate novel therapeutic agents to control I. multifiliis.
Nanoparticles with a diameter ⩽100 nm are now being increasingly applied for medical purposes and have attracted immense attention as an alternative approach to control infectious agents (Swain et al. 2014). Nanoparticles are characterized by their large surface area and high particle number per unit mass compared with bulk materials (Buzea et al. 2007). They have unique physical, chemical, and biological characteristics, which are currently the subject of much scientific research. In aquaculture, metal and metal oxide nanoparticles exhibit effective antimicrobial properties against fish pathogens (Swain et al. 2014), and have been utilized in water decontamination and as antimicrobial agents Parasitology (2017), 144, 1802-1810. © Cambridge University Press 2017. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi:10.1017/S0031182017001184 (Li et al. 2008;Rana & Kalaichelvan, 2011;Saleh et al. 2016;Shaalan et al. 2016). Due to their nontoxicity, high ability for functionalization and polyvalent effects, gold nanoparticles are promising in the development of novel antimicrobial agents (Tiwari et al. 2011;Zhou et al. 2012;Lima et al. 2013;Lolina & Narayanan, 2013;Saleh et al. 2016). The antimicrobial activity of the nanoparticles is attributed to their attachment to microbial cell membranes, followed by alteration of membrane potential, decrease of ATP level and inhibition of tRNA binding to the ribosome (Cui et al. 2012). However, particles can aggregate when weakly bound capping agents like citrate are used, which leads to reduced surface area and decreased interactions with the nanoparticles (Zhou et al. 2012). In contrast, gold nanoparticles with the same shape and size but capped with strongly bound agent may exhibit enhanced antimicrobial properties (Zhou et al. 2012;Dizaj et al. 2014). The antimicrobial activity of zinc oxide nanoparticles is due to contact between nanoparticles and pathogen cells, which alters surface charges and electrostatic interactions (Stoimenov et al. 2002;Neal, 2008;Zhang et al. 2010), leading to rupture of the cell membrane (Zhang et al. 2007;Jiang et al. 2009). In aquaculture, zinc oxide nanoparticles have been reported to affect the growth of Aermonas hydrophila, Edwardseilla tarda, Flavobacterium branchiophilum, Citrobacter species, Staphylococcus aureus, Vibrio species, Bacillus cereus and Pseudomonas aeruginosa (Ramamoorthy et al. 2013;Swain et al. 2014).
Silver nanoparticles have been reported to affect microorganisms by different mechanisms (Franci et al. 2015). Silver nanoparticles bind to cell membrane proteins and disrupt cell membrane and induce production of reactive oxygen species leading to cell death (Lara et al. 2010). Intracellulary, they bind to cytochrome and interfere with nucleic acids of the pathogens, thereby inhibiting cell division and subsequent replication (Lara et al. 2010;Shaalan et al. 2016). Silver nanoparticles have been shown to affect morphology and pathogenicity of the protozoan parasite Leishmania tropica in vitro (Allahverdiyev et al. 2011).
Given the versatile characteristics and low toxicity of nanoparticles, we consider they represent a potentially useful approach to manage pathogens in aquaculture. In this study, we investigated the effects of gold, silver and zinc oxide nanoparticles on survival and reproduction of I. multifiliis free-living stages, and assessed the infectivity of surviving theronts to rainbow trout in vivo.

Fish and parasite
The life cycle of I. multifiliis in the laboratory was initiated by cohabitation of juvenile rainbow trout (Oncorhynchus mykiss) (∼5 g) obtained from a registered disease free commercial fish farm in Vienna, Austria, with naturally infected common carp (Cyprinus carpio) acquired from carp pond. The infected carp were kept with 10 rainbow trout in a 250 L tank for 7 day to allow infection of the rainbow trout by I. multifiliis. The temperature of the water was controlled at 16 ± 2°C, and the fish were fed daily at 1% of fish weight. When rainbow trout were heavily infected with mature trophonts, they were anesthetized (150 mg L −1 tricaine methanesulfonate (MS-222, Sigma). Subsequently, skin was gently scraped into Petri dishes that contained 10 mL of water at 16°C, and trophonts were allowed to escape the fish mucus. The free-swimming trophonts (protomonts) were collected and rinsed several times with dechlorinated water to eliminate residual fish mucus. All experiments were approved by the Animal Experimentation Ethics Committee of Vienna University of Veterinary medicine (BMWFW-68·205/0051-WF/ V/3b/2016).

Collection of the parasite stages
Protomonts were collected and placed in batches of 50 in Petri dishes containing 10 mL filtered freshwater and either used directly or incubated at 15 ± 1°C, until they reached either the encysted tomont stage (minimum eight cells) or the theront stage (after ∼23-30 h). To obtain tomonts, the Petri dishes with trophonts were incubated for only 16 h at 15°C so that the development of theronts was not complete. To obtain theronts, the protomonts were put into Petri dishes (approximately 50 trophonts per Petri dish) with 10 mL of dechlorinated water and incubated at 15°C for 24 h, after which time theronts were released. To determine the number of theronts produced, 10 × 20 µL subsamples were put on slides fixed with 5 µL Roti-Histofix ® (Carl Roth) and counted under a microscope; the mean count was used to assess the total number of theronts produced. A dual fluorescent staining technique using propidium iodide and fluorescein diacetate was used to differentiate between viable and damaged parasites by fluorescent microscopy (Schumacher et al. 2011).

Zinc oxide nanoparticles
The zinc oxide nanoparticles (∼66 nm) were purchased from Sigma Aldrich, Austria, together with all reagents used for in-house synthesis of gold and silver nanoparticles.

Gold nanoparticles
We synthesized gold nanoparticles by reduction of tetrachloroauric acid (HAuCl 4 ) with sodium citrate . Briefly, an aqueous solution of HAuCl 4 ·3H 2 O was brought to boil under reflux with stirring. After rapid addition of 10 mL of 1% trisodium citrate, the colour of the solution changed from yellow to deep red. After an additional 15 min reflux, the solution was allowed to cool to room temperature, before being filtered through a 0·45 µm acetate filter, and then stored at 4°C.

Silver nanoparticles
We synthesized silver nanoparticles by chemical reduction of silver nitrate according to the protocol of El Mahdy et al. (2015). Both sodium citrate tribasic hydrate and sodium borohydride were utilized as reducing agents, and polyvinyl pyrrolidone as a stabilizing agent to prevent particle agglomeration (Wang et al. 2005). The solution was stored in an autoclaved, dark bottle at 4°C.

Characterization of nanoparticles
Formation of gold and silver nanoparticles was confirmed by Ultraviolet-visible spectral analysis. The absorbance spectra were recorded using (NanoDrop 2000 ® ). Deioinized water was used as a blank. All measurements were performed at room temperature on three different days. The morphology of the synthesized gold and silver nanoparticles was investigated using TEM (EM 900, Zeiss, Oberkochen, Germany) and Image SP Viewer ® software was used to calculate their mean size from 100 randomly sampled nanoparticles. We used a Zetasizer Nano ZS ® (Malvern.com), to measure size distribution of the nanoparticles based on dynamic light scattering (DLS). Triplicate measurements were performed at room temperature.
In vitro exposure of protomonts to gold, silver and zinc oxide nanoparticles Approximately 50 protomonts in 500 µL dechlorinated freshwater were placed into each well of a 24-well tissue culture plate. 500 µL of each nanoparticle solution (gold, silver and zinc) was added in triplicates, to attain final concentrations of 0 (control), 2·5, 5·0, 10·0, 20·0, 40·0, 80·0 and 160 ng mL −1 . The efficacy of each dose was assessed by counting the number of protomonts at 15 and 30 min, 1, 2, 4, 6, 12, 18 and 24 h until the protomonts had either died or theronts were released. Protomonts were classified as active (survival) or motionless (dead) using a microscope at 40 × or by using the dual fluorescent staining technique as described above. The numbers of encysted tomonts and released theronts were determined at 6 and 24 h post-exposure to nanoparticles, respectively.
In vitro exposure of encysted tomonts to gold, silver and zinc oxide nanoparticles Approximately 30 encysted tomonts in 500 µL dechlorinated freshwater were placed into each well of a 24-well tissue culture plate. Only 30 encysted tomonts were used because not all of the protomonts encysted successfully. 500 µL of each nanoparticle solution was added in triplicates, to final concentrations of 160, 80, 40, 20, 10, 5, 2·5 and 0 ng mL −1 . Encysted tomonts were categorized as active (survival) or motionless (dead) as above. The numbers of released theronts were determined at 24 h postexposure to nanoparticles.
In vitro exposure of I. multifiliis theronts to gold, silver and zinc oxide nanoparticles Wells of 24-well plates in triplicates were loaded with 500 µL theront suspension containing ∼ 150 theronts enumerated as described above. Then, 500 µL of each nanoparticle solution was added to wells to reach final concentrations of 160, 80, 40, 20, 10, 5, 2·5 and 0 ng mL −1 . The number of theronts surviving in each well was determined at 15 and 30 min, 1, 2, 4, 6, 12, 18 and 24 h post exposure. Triplicates were set up for each concentration and each time point, each triplicate originating from the same group of parasites to reduce possible different survival rates among different cohorts.
The per cent inhibition of I. multifiliis free-living stages was calculated as per cent inhibition = 100−[(mean number of viable parasites counted in exposed wells/mean number of parasites counted in non-exposed wells) × 100]. The differences between nanoparticles exposed and non-exposed parasites were analysed using t tests with Bonferroni α-correction. For all statistical tests, a P value <0·05 was regarded as significant. Statistical analyses were conducted with SPSS V.20 software.
The ability of theronts surviving exposure to subsequently infect fish To determine whether theronts treated with nanoparticles had the ability to infect fish, nine Petri dishes (three triplicates) were prepared, each containing 25 mL of theronts suspension (∼150 theronts mL −1 ) drawn from one pool of theronts. Subsequently, 25 mL of silver, zinc oxide nanoparticles or filtered freshwater (control) were added to each dish to a final volume of 50 mL and a final concentration of 10 (silver), 5 (zinc) or 0 (control) ng mL −1 nanoparticles. Dishes were incubated at 15°C for 18 h.
The number of live vs dead theronts in three separate 1 mL aliquots taken from each Petri dish was determined. The remaining 47 mL was then added to separate tanks of rainbow trout to determine if any surviving theronts could infect fish.
For the infection trial, triplicate 10 L tanks were maintained in a constant temperature at 15°C. Each tank contained 10 ∼5 g O. mykiss fingerlings. The fish in each tank were exposed to the relevant batches of theronts for 3 h under static conditions, in the dark with aeration.
Fish were maintained for 10 days at 15°C on a 2% body weight day −1 ration of commercial feed. Fish were then killed using an overdose of 150 mg L −1 tricaine methanesulfonate (MS-222, Sigma, Austria). The total number of trophonts on the fins, gills and entire body surface was recorded.
The ability of trophonts collected from fish after infection with theronts surviving zinc nanoparticles exposure to subsequently encyst into tomonts and release theronts Trophonts were collected from infected fish 10 day post-exposure (from above) and placed into Petri dishes, which contained 10 mL water at 15°C. Their ability to encyst into tomonts and release theronts was observed at 6 and 24 h, respectively.

R E S U L T S
UV-Vis analysis of gold and silver nanoparticles showed maximum absorption at 523 and 395 nm, which matched expected values for gold and silver nanoparticles, respectively. TEM revealed both gold and silver nanoparticles to be spherical, with mean diameters of 18 and 21 nm, respectively. DLS measurements showed one peak at 23 nm for gold nanoparticles, and two peaks (at 8·3 and 44·5 nm) for silver nanoparticles. Parasite stages were categorized as active (survival, Fig. 1A, C, E) or motionless (dead, Fig. 1B, D, F). For samples where the dual fluorescence staining technique was used, intact parasites fluoresced green, while dead or damaged stages fluoresced red.
Protomonts: Exposures to 20, 10, 5 ng mL −1 gold, silver or zinc oxide nanoparticles for 30 min resulted in ∼50% mortality (Table 1). About half of the protomonts stopped moving after exposure, and displayed slow ciliary movement with some developing small projections over their surface and rupturing after 30 min (Fig. 1B, D). The surviving protomonts showed asymmetric division, and released theronts after 72 h. Negative control protomonts encysted successfully and released theronts after ∼24 h. Exposure to doses lower than 5 ng mL −1 for 30 min were less successful in killing I. multifiliis protomonts, however, longer exposure time (⩾2 h) increased protomont mortality (Table 1).
Encysted tomonts: Approximately 50% of tomonts exposed to 20, 10, 5 ng mL −1 gold, silver or zinc oxide nanoparticles were killed by a 30 min exposure, and for survivors, speed of development was affected, and subsequent release of theronts was delayed. Exposure to doses lower than 5 ng mL −1 was less effective, and tomonts released theronts after ∼24 h, similar to the control unexposed encysted tomonts. Exposure of encysted tomonts to 20, 10, 5 ng mL −1 gold, silver and zinc oxide nanoparticles for 2 h resulted in 100% mortality (Table 1).
In vitro exposure of I. multifiliis theronts: survival of theronts after exposure to 20, 10, 5 ng mL −1 gold, silver and zinc oxide nanoparticles for up to 24 h demonstrated a dose and time dependent response, with survival decreasing with increased concentrations of nanoparticles (Table 1). However, increasing concentration above the 20, 10, 5 ng mL −1 gold, silver and zinc oxide nanoparticles did not affect mortality significantly. With 10 ng mL −1 silver nanoparticles, theront survival decreased with exposure time. After 2 h, no theronts survived in the silver nanoparticle exposure group, compared with 100% survival in the control; after 24 h, all exposed theronts had died. Theront survival after exposure to 5 ng mL −1 zinc oxide nanoparticles also showed a reduction over time, however, after 24 h ∼3% of theronts still survived.
In vivo fish-infection trials: Fish cohabited with control theronts (no nanoparticle exposure) had 100% mortality, with a mean of 110 ± 20 trophonts attached to the skin of each fish ( Fig. 2A). After 24 h, no theronts survived exposure to silver nanoparticles, and no infections were recorded in fish cohabited with this group (Fig. 2B). Fish cohabited with the theronts after exposure to zinc oxide nanoparticles became infected, with 1-7 trophonts attached to each fish (Fig. 2C-F). These trophonts were collected, then failed to successively encyst and release theronts. However, the infection level in this group was significantly lower than that observed in the control group. No mortalities were recorded in the fish groups exposed to either silver or zinc oxide nanoparticles.

D I S C U S S I O N
Due to the complex life cycle of I. multifiliis and the scarceness of effective treatments, novel therapeutics are being investigated for disease management (Dickerson and Findly, 2014). Some success against aquatic fish pathogens has been shown with therapeutic strategies that utilize metal nanoparticles, specifically gold and silver nanoparticles (Soltani et al. 2009;Vaseeharan et al. 2010;Umashankari et al. 2012;Antony et al. 2013;Mahanty et al. 2013;Velmurugan et al. 2014;Saleh et al. 2016;Shaalan et al. 2016) and zinc oxide nanoparticles (Ramamoorthy et al. 2013;Swain et al. 2014). Based on these results, in the present study we investigated the antiprotozoal activity of gold, silver and zinc oxide nanoparticles against free-living stages of I. multifiliis.
Exposure of the free-living theront stages of I. multifiliis to 20, 10 and 5 ng mL −1 gold, silver and oxide nanoparticles killed 48, 52 and 50% of the parasites, respectively, and ∼50% of both protomonts and encysted tomonts after 30 min exposure. Protomonts surviving exposures successfully transformed into encysted tomonts; however, these tomonts showed asymmetric cell division and/or delayed development time to release theronts (72 h compared with 24 h for control). Exposure of encysted tomonts to the same concentrations of nanoparticles killed 50% within 30 min and the rest over the subsequent 48 h. Lower concentrations of nanoparticles were less effective in killing encysted tomonts, although they affected the parasite metabolism and delayed theronts release.
We observed significantly higher survival (∼22%) of theronts after exposure to ⩾20 ng mL −1 gold nanoparticles than to silver and zinc oxide nanoparticles, and attributed this to higher aggregation of the gold nanoparticles due to the weakly bound capping citrates used in the synthesis (Zhou et al. 2012), and thus gold nanoparticles were not used in subsequent in vivo trials. We found using in vitro assays that zinc oxide nanoparticles reduced the number of theronts over time, however, those that survived exposure were still able to infect fish.
Previous work has shown silver nanoparticles to be effective against I. multifiliis trophonts using a bath exposure at 10 ng g −1 body weight of silver nanoparticles, which left fish infection free for more than 12 months (Daniel et al. 2016). However, prior to our current study, no assessment had been made with silver nanoparticles on the  different free-living developmental stages of I. multifiliis, which are considered major factors for emergence and spread of the disease. We found that silver nanoparticles killed all theronts in vitro, no fish became infected by the end of the experiment, and none died within 10 days (the end of the trial). In contrast, all fish in the control group became heavily infected and died by the end of the experiment. Silver nanoparticles are reported non-cytotoxic (Arora et al. 2008), however at higher concentrations, rainbow trout hepatocytes cells can be affected (Farkas et al. 2010). Also, cytotoxic and genotoxic effects have been observed on fish cell lines and zebrafish at higher concentrations of silver nanoparticles (Kim & Ryu, 2013). We have observed that a concentration of 16 µg mL −1 did not affect the viability of EK-1 cells assessed using an MTT assay, and silver nanoparticles did not cause mortality (within the 30-day experiment) in rainbow trout exposed via either an immersion bath at 100 µg L −1 , or intra peritoneal injection (1 µg g −1 ) (El-Matbouli, unpublished data).
In the present study, we demonstrated that 10 ng mL −1 silver nanoparticles were effective against all I. multifiliis free-living stages, and did not appear to be cytotoxic towards the fish. The precise mechanism by which silver nanoparticles exhibit their action is unknown, but likely involves disruption of the I. multifiliis membranes, which would lead to release of immature theronts from encysted tomonts, which are unable to infect fish.
In conclusion, we demonstrated that while all tested nanoparticles inhibited the development of I. multifiilis, silver nanoparticles were most effective. Silver nanoparticles adversely affect the survival of all free-living stages of I. multifiliis (protomonts, tomont and theronts) and could be applied in disease management. However, we propose future investigations aimed at increasing the specificity of nanoparticles towards particular pathogens, through modification of the nanoparticle surfaces by conjugation with ssRNA or siRNA that binds to known pathogen sequences (silencing/knocking down the target gene). This could lead to Fig. 2. Fish exposed to theronts that had been incubated for 24 h with silver nanoparticles did not become infected, whereas the control fish group showed 100 and 50% infection and mortality rates, respectively ( Fig. 2A, B). Theronts survived exposure to zinc oxide nanoparticles were able to infect 50% of the fish, but at a low intensity (1-7 attached trophonts) (Fig. 2C-F). pathogen-specific nanoparticle treatments for disease management in aquaculture.

A C K N O W L E D G E M E N T S
We gratefully acknowledge the assistance of M. Shaalan and would like to thank K. Pichler for feeding and taking care of the fish. F I N A N C I A L S U P P O R T This work was supported by the national plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz city for science and technology, Kingdom of Saudi Arabia.