Saleh et al. BMC Veterinary Research (2016) 12:44 DOI 10.1186/s12917-016-0668-x RESEARCHARTICLE Open Access In vitro antimicrosporidial activity of gold nanoparticles against Heterosporis saurida Mona Saleh1, Gokhlesh Kumar1, Abdel-Azeem Abdel-Baki2,3, Saleh Al-Quraishy2 and Mansour El-Matbouli1* Abstract Background: Worldwide, there is a need to expand the number of drugs available to treat parasitic infections in aquaculture. One of the new materials being tested is metal nanoparticles, which have unique chemical and physical characteristics owing to their extremely small size and high surface area to volume ratio. We examined the effectiveness of gold nanoparticles against the microsporidian parasite Heterosporis saurida, which causes severe economic losses in lizard fish, Saurida undosquamis aquaculture. Results: We synthesized gold nanoparticles by chemical reduction of tetrachloroauric acid as a metal precursor. We assessed the antimicrosporidial efficacy of the nanoparticles against H. saurida using an in vitro screening approach, which we had developed previously using the eel kidney cell line EK-1. The number of H. saurida spores produced in EK-1 cells was reduced in a proportional manner to the dosage of gold nanoparticles administered. A cell metabolic activity test (MTT) indicated that the gold nanoparticles did not appear to be toxic to the host cells. Conclusions: Gold nanoparticles can act as an effective antimicrosporidial agent and hold promise to reduce disease in lizardfish aquaculture. Metal nanoparticles should be considered as an alternate choice for development of new antimicrosporidial drugs to combat disease problems in aquaculture. Keywords: Gold nanoparticles, Microsporidia, Heterosporis saurida, Lizardfish, EK-1 cells Background Microsporidia are single-celled, obligate intracellular Saurida undosquamis (Richardson, 1848) also known as parasites that infect invertebrates and vertebrates. More Brushtooth lizardfish is fish species of the Synodontidae than one thousand microsporidian species have been family. S. undosquamis is a Lessepsian migrant species recognized as causing diseases in animals and humans distributed across the Indo-West Pacific including the [7]. Microsporidia are common in marine, fresh water Red Sea, Persian Gulf, Eastern Africa, Japan and and estuarine systems, and affect economically import- Australia [1]. S. undosquamis invaded the Levant Basin ant fish species worldwide, including salmonids [8], flat- of the Mediterranean Sea, from the Indo-West Pacific fish [9], greater sand eels (Hyperoplus lanceolatus) [10] through the Suez Canal [2] and is considered one of the and ornamental fish, such as zebrafish (Danio rerio) [11] most successful colonizers of the Eastern Mediterranean, and killifish (Family Cyprinodontidae) [12]. Infections extending as far as the Aegean Sea [3]. The Mediterranean caused by microsporidia reduce the growth rate of fish lizardfish population now has significant commercial value and decrease production in aquaculture [13, 14]. Re- [4] in the eastern Mediterranean, where it is considered cently, H. saurida was isolated from lizardfish (Fig. 1) one of the most common species caught in the trawl where it infects skeletal muscle, body cavity and mesen- fishery [5]. teric tissues and forms white, cyst-like structures, which Diseases are a major obstruction to expansion of fresh contain numerous spores making the fish unsuitable for water and marine aquaculture. Fish are susceptible to public consumption [15]. many pathogens, often with severe consequences [6]. Only a small number of anti-parasitic drugs are per- mitted in aquaculture, as the cost of drug development * Correspondence: [email protected] is high and the market small. The few available treat- 1 Clinical Division of Fish Medicine, University of Veterinary Medicine, ments for microsporidiosis differ in their effectiveness, Veterinaerplatz 1, 1210 Vienna, Austria Full list of author information is available at the end of the article and as with many drug treatments, there is concern for © 2016 Saleh et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Saleh et al. BMC Veterinary Research (2016) 12:44 Page 2 of 6 saurida, in vitro propagation has been demonstrated for both mammal (rabbit kidney; [34]) and fish (eel kidney EK-1; [35]) cell lines and an in vitro screening approach using the EK-1 cells to test antimicrosporidial agents against the parasite was also developed [36], however, our aim in the present study was to investigate the in vitro antimicrosporidial activity of gold nanoparticles as a potential agent against H. saurida infections. Methods Fig. 1 The microsporidian parasite Heterosporis saurida infects the lizard fish Saurida undosquamis and forms white, cyst-like structures Ethics statement in the muscles No ethical approval was necessary as this study did not involve laboratory animals; it comprised in vitro testing of cultured cell lines and microsporidian spores. the pathogens developing resistance to one or multiple antibiotics [16]. A promising, alternative approach that Propagation of H. saurida spores has received recent attention is the use of metallic nano- Heterosporis saurida spores were grown in the eel fish particles, which have distinct advantages over conven- kidney cell line (EK-1) using procedures previously de- tional antimicrobial agents [17, 18]. Nanometer-sized scribed [35]. Briefly, H. saurida spores were collected materials have unique, extraordinary and fascinating phys- from naturally infected lizard fish, Saurida undosquamis ical, chemical, and biological properties, particularly a [15]. EK-1 cells were sub-cultured, seeded in 24-well large contact area with microorganisms because of their plates in triplicate and supplemented with L-15 medium small size and higher surface-to-volume ratio; a property (Leibovitz) containing L-glutamine (Sigma-Aldrich), 10 % that expands their biological and chemical activity. Cur- fetal bovine serum (FBS) (Sigma-Aldrich), 100 units/ml rently, nanoparticles with one dimension of 100 nm or penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, less have received immense interest as an alternative Vienna, Austria). Twenty four hours after seeding, 100 μl means of combatting infectious agents in medicine. Metal (~107 spores/ml) of spore suspension was added to each based nanoparticles display broad spectrum antimicrobial well. Twenty-four hour post-inoculation (p.i.), the medium activity against bacteria, fungi and viruses [19–24]. was removed and the cells rinsed gently 3 times with fresh The most widespread antimicrobial compounds are medium; the medium was replace twice per week. silver and benzalkonium chloride [25, 26]. Unfortu- nately, in a comparable manner where scientists develop Preparation and characterization of gold nanoparticles new efficient antimicrobial materials, there is no doubt We followed the protocol of Storhoff et al. [37] to that resistance to silver also is increasing [16]. Therefore, synthesize gold nanoparticles (~13 nm diameter) by re- a number of metals, mainly copper, have been used as duction of 10 mM tetrachloroauric acid (HAuCl4) using an antimicrobial agent. Gold has been little explored as sodium citrate (Sigma-Aldrich, Vienna, Austria). Briefly, an antimicrobial agent, although it has been used as a an aqueous solution of HAuCl4.3H2O was boiled under catalyst [27, 28]. reflux while being stirred. The color of the solution Gold nanoparticles in particular, have a broad range of changed from yellow to deep red after rapid addition of applications in nano-scale devices and technologies due 10 ml 1 % trisodium citrate. The color change signified to their chemical inertness and resistance to surface oxi- formation of monodispersed spherical gold nanoparti- dation [29]. Gold nanoparticles have been investigated as cles. The solution refluxed for an additional 15 min, then an antimicrobial agent to inhibit the growth of common, allowed to cool to room temperature. The solution was waterborne, pathogens Escherichia coli and Salmonella subsequently filtered through a 0.45 μm acetate filter typhi, which are developing resistance to common bacte- and stored at 4 °C. A stock concentration of 1 mg/ml ricides [30]. In aquaculture, several studies have been gold nanoparticles was used in the assays. carried out to investigate gold nanoparticles as anti- Morphology, size, and shape of synthesized gold nano- microbial drugs [31–33]. particles were characterized using transmission electron In vivo efficiency trials for anti-parasitic drugs are ex- microscopy (TEM). Samples were prepared by drop cast- pensive, time intensive, and need ethical permission, ing a 2.5 mL aliquot of the Au NPs suspension onto a hence new and efficient systems to screen compounds 300 mesh carbon-coated copper grid. The gold suspen- while reducing animal experiments are required. One sion was dried at room temperature for 5 min and over- alternative, cost-effective approach is to