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ARTICLE IN PRESS International Journal of Antimicrobial Agents xxx (2015) xxx–xxx

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The first comprehensive study of praziquantel effects in vivo and in vitro on European liver fluke Opisthorchis felineus (Trematoda) Mariya Y. Pakharukova a,b,∗ , Alexander G. Shilov a , Darya S. Pirozhkova a , Alexey V. Katokhin a , Viatcheslav A. Mordvinov a,c a

Institute of Cytology and Genetics (ICG), Siberian Branch of the Russian Academy of Sciences (SB RAS), 10 Lavrentiev Ave., Novosibirsk 630090, Russia Novosibirsk State University, 2 Pirogov Street, Novosibirsk 630060, Russia c Institute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Sciences (SB RAMS), Tymakova 2, Novosibirsk 630060, Russia b

a r t i c l e

i n f o

Article history: Received 3 July 2014 Accepted 12 February 2015 Keywords: Praziquantel Opisthorchis felineus Surface damage Worm reduction

a b s t r a c t The European liver fluke Opisthorchis felineus (Rivolta, 1884) is an epidemiologically important parasite infecting mammals, including humans. Opisthorchis felineus is widespread in Russia, Kazakhstan and Eastern European countries. Praziquantel (PZQ) is the drug of choice for the treatment of opisthorchiasis, but the effects of this drug on O. felineus are poorly studied. The aims of this work were (i) to perform a study of PZQ effects in vitro, (ii) to identify morphological markers of PZQ action on O. felineus, (iii) to analyse damage to the worm surface and (iv) to assess the efficacy of PZQ in vivo in a hamster model. Light microscopy, optical sectioning and fluorescence microscopy were used to study morphological changes. In vivo, PZQ at a dose of 400 mg/kg reduced the rate of infection in experimental acute and chronic opisthorchiasis in hamsters by 70% and 79%, respectively. In vitro, the drug caused destruction and vacuolisation of the tegument of O. felineus, contractions of the worm musculature, paralysis, and irreversible changes in morphology (IC50 = 0.14 ␮g/mL). Differences in susceptibility to the drug between adult and newly excysted metacercariae were also observed. Qualitative effects of PZQ in vivo and in vitro were similar to the drug’s effects on other trematodes, including epidemiologically important liver flukes. Nevertheless, high heterogeneity of O. felineus specimens in terms of susceptibility to the drug was observed. In addition, we describe for the first time the high rate of recovery of O. felineus following the destructive action of PZQ. © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction Opisthorchiasis is an important food-borne trematodiasis that is highly prevalent in the former Union of Soviet Socialist Republics (USSR) and Eastern European countries [1–3]. In particular, the ObIrtysh basin is thought to be the world’s largest endemic centre of opisthorchiasis caused by Opisthorchis felineus (Rivolta, 1884) (Opisthorchioidea, Trematoda, Platyhelminthes). Recent studies have assessed the prevalence of O. felineus in Europe (reviewed in [2]) and these flukes were found on the Iberian Peninsula, the Balkan Peninsula, in Germany and in Italy [4]. The estimated infection rate of O. felineus is 1.6 million of 17 million opisthorchiatic patients worldwide [5].

∗ Corresponding author at: Institute of Cytology and Genetics (ICG), Siberian Branch of the Russian Academy of Sciences (SB RAS), 10 Lavrentiev Ave., Novosibirsk 630090, Russia. Tel.: +7 913 394 66 69; fax: +7 383 333 12 78. E-mail address: [email protected] (M.Y. Pakharukova).

At present, praziquantel (PZQ) is the anthelmintic drug of choice and is used for the treatment of opisthorchiasis, clonorchiasis, schistosomiasis and other diseases caused by trematodes. In many endemic regions of the world, PZQ is used in preventive chemotherapy programmes against parasitic infections [6]. PZQ causes immobilisation, spasmodic contractions, paralysis and tegument damage as evidenced by extensive swelling, erosion, vacuolisation and peeling. In this context, O. felineus has failed to attract much attention from researchers. Despite the epidemiological significance of this parasite, there are few studies on the effects of PZQ in O. felineus. In the published studies, the focus of attention is usually on the therapeutic properties of this drug. In particular, there are several reports about the cure rates of opisthorchiasis treatment with PZQ in humans in different parts of Russia [3,7,8] as well as in some case studies of dogs [9]. To our knowledge, a comprehensive study of the effects of PZQ on O. felineus in vivo and in vitro has not yet been published. Opisthorchis felineus is a typical trematode and has a complex life cycle, including two intermediate hosts and one definitive (final)

http://dx.doi.org/10.1016/j.ijantimicag.2015.02.012 0924-8579/© 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Please cite this article in press as: Pakharukova MY, et al. The first comprehensive study of praziquantel effects in vivo and in vitro on European liver fluke Opisthorchis felineus (Trematoda). Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2015.02.012

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ARTICLE IN PRESS M.Y. Pakharukova et al. / International Journal of Antimicrobial Agents xxx (2015) xxx–xxx

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host (mammals). Rodents, including mice, rats and hamsters, are an accepted laboratory model of parasitoses [10,11]. In the present work, the action of PZQ was studied in an experimental model of opisthorchiasis in hamsters. The aims of this work were: (i) to study the action of PZQ at a wide range of concentrations in vitro; (ii) to conduct a comparative analysis of the effects of PZQ at different stages of the life cycle; (iii) to identify morphological markers of PZQ action on O. felineus; (iv) to analyse damage to the worm morphology; and (v) to assess the efficacy of this drug in vivo. 2. Methods 2.1. Animals and parasites Golden Syrian hamsters (Mesocricetus auratus) were purchased from the stock of the Pushchino Animal Facility (Pushchino, Moscow Region, Russia) and were bred at the Animal Facility of the Institute of Cytology and Genetics (ICG), Siberian Branch of the Russian Academy of Sciences (SB RAS) (Novosibirsk, Russia). All of the procedures were in compliance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for animal experiments (http://www.ec.europa.eu/environment/ chemicals/lab animals/legislation en.htm). Animals were kept and treated according to protocols approved by the Committee on the Ethics of Animal Experiments of the ICG. Euthanasia was performed by decapitation and all efforts were made to minimise suffering. Opisthorchis felineus metacercariae were collected from naturally infected fish (Leuciscus idus) caught in the Ob River near the city of Novosibirsk (Western Siberia). Territories where sample collection (fishing) took place were neither conservation areas nor private, nor otherwise protected; hence, no fishing permits were required. The fish species collected are not considered endangered or rare, and the fishing methods were in full compliance with the Federal Law N166-F3 of 20.12.2004 (ed. 18.07.2011) ‘Fishing and conservation of water bio-resources’.

under an inverted microscope (Axiovert 40CFL) equipped with an AxioCam ICc3 (Zeiss, Oberkochen, Germany) after 1, 4 and 24 h of treatment (magnification 10–50×). Worms were classified as dead if they had a dark colour and no movement was observed for 2 min [13]. After 24 h, the worms were washed with sterile saline several times and were placed in the medium without drug. Parasites were cultivated under the same conditions for 24–48 h after removal of the drug. Viability of the worms was assessed again as described above. For calculation of the IC50 (defined as the concentration of drug required to the decrease the mean worm’s motility to 50% at the 24-h time point), PZQ was tested at concentrations of 0.001, 0.01, 0.1, 0.25, 0.5 and 1 ␮g/mL. Ten adult worms were used per concentration. The motility of viable worms was assessed on a scale of 0 to 3 ([+++] very active (similar movement to the control flukes); [++] active (reduced motility compared with the controls; however, the entire body was still moving); [+] reduced viability (only movements of the oral sucker); and [−] non-motile) [14]. The mean motility score for PZQ was calculated for each concentration and was normalised as a percentage relative to the control. The IC50 value was expressed using CompuSyn software v.1.0 [15]. 2.4. Assessment of surface damage A propidium iodide (PI) assay was used for evaluation of tegument damage. PI was added to the medium containing the drug just before microscopy (20–30 min in advance). After 20 min, worms were washed in saline and were placed on a microscope slide in a DABCO solution [0.5% DABCO (Sigma-Aldrich), 90% glycerol (Sigma-Aldrich) and 10% phosphate-buffered saline (PBS), pH 7.6]. The slides were examined with rhodamine (536 nm excitation) and fluorescein isothiocyanate (FITC) (494 nm excitation) filters under an Axio Imager fluorescence microscope with ApoTome (Zeiss). Images were processed with AxioVision software (Zeiss). As a control, live and heat-killed worms stained with the same concentration of PI (2 ␮g/mL) were used [16]. Heat-killed worms were prepared by incubating the parasites at 65 ◦ C for 10 min.

2.2. Drugs 2.5. In vivo study PZQ (Bayer, Leverkusen, Germany) for in vitro experiments was dissolved in dimethyl sulphoxide (DMSO) (Sigma-Aldrich, St. Louis, MO) to obtain a stock solution of 10 mg/mL. PZQ was added as 100× solution to achieve final concentrations of 0.1–100 ␮g/mL. Controls received an equivalent amount of vehicle (DMSO). For in vivo studies, PZQ was prepared as a suspension in 7% Tween 80 (v/v) and 3% ethanol (v/v) before oral administration (10 mL/kg). Propidium iodide (Sigma-Aldrich) was dissolved in water to prepare a 1 mM stock solution. 2.3. In vitro study Newly excysted metacercariae (NEM) were hatched from metacercariae [12]. Adult worms were grown by infecting golden hamsters with metacercariae (100 specimens per individual) [12]. Hamsters were killed 3 months after inoculation. Flukes were recovered from the livers of untreated hamsters and were thoroughly washed with sterile saline (0.9% NaCl). PZQ was serially diluted in 24-well plates (Corning® Costar® ; Sigma-Aldrich) in RPMI 1640 culture medium (Life Technologies, Carlsbad, CA) in a CO2 incubator (in a humidified atmosphere of 5% CO2 ). PZQ was tested at concentrations of 0.1, 1, 10 and 50 ␮g/mL. Each concentration was assessed in duplicate and the experiment was repeated twice. A total of 20–30 adult worms and 70–80 NEM were used for each concentration. Worms were incubated at 37 ◦ C for 24 h with the drug. Their viability was evaluated several times

For in vivo experiments, PZQ was used as a suspension in 7% Tween 80 and 3% ethanol before oral administration (10 mL/kg). Untreated animals served as controls [14]. Two schemes were chosen for PZQ treatment in vivo. The first scheme was based on the treatment of chronic experimental opisthorchiasis [17]. Twenty-three hamsters infected by 100 metacercariae 3 months before the experiment were selected. The hamsters were divided into three groups. Six animals received 75 mg/kg PZQ orally three times a day; seven animals received 400 mg/kg PZQ orally once [14]; and eleven untreated animals served as controls [14]. The second scheme was based on the use of PZQ treatment of acute experimental opisthorchiasis, with animals infected 1 month before the experiment. Eleven hamsters were infected with 100 metacercariae. The hamsters were divided into two groups. One group (six animals) received 400 mg/kg PZQ orally once [14] and five untreated animals served as controls [14]. After 21 days from the beginning of the study, animals were killed and necropsied. Mann–Whitney U-test was used to assess the differences between the means. Differences with P values of