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Fasciola hepatica: Surface tegumental responses to in vitro and in vivo treatment with the experimental fasciolicide OZ78
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Experimental Parasitology 119 (2008) 87–93 www.elsevier.com/locate/yexpr
Fasciola hepatica: Surface tegumental responses to in vitro and in vivo treatment with the experimental fasciolicide OZ78 Jennifer Keiser a,*, Gianni Morson b b
a Swiss Tropical Institute, Medical Parasitology and Infection Biology, Socinstr. 57, P.O. Box, CH–4002 Basel, Switzerland Center for Microscopy, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, University of Basel, Switzerland
Received 30 October 2007; received in revised form 17 December 2007; accepted 30 December 2007 Available online 8 January 2008
Abstract The tegumental alterations in adult Fasciola hepatica induced by the experimental fasciolide OZ78 were investigated utilizing scanning electron microscopy (SEM). Twelve weeks post-infection with F. hepatica, rats were treated with a single 100 mg/kg oral dose of OZ78 and flukes were recovered from the bile ducts after 24–72 h. In vitro F. hepatica were incubated with OZ78 for 48 h at a concentration of 10 lg/ml in the absence or presence of haemin. Twenty-four and 48 h post-treatment of rats disruption of the tegument of F. hepatica as blebbing, swelling and furrowing was evident. The recovery of flukes 72 h post-treatment was low. Flukes examined at this time point showed an increasing severity of tegumental damage as sloughing and absence of spines, in particular in the tail region. SEM analysis of F. hepatica incubated in the presence of OZ78 without haemin showed only minor and localized damage of the tegument. In the presence of haemin extensive tegumental damage, including sloughing or blebbing, in particular in the anterior part, was observed. In conclusion, our experiments confirm the interesting fasciocidal properties of OZ78. The tegument of adult F. hepatica might play a role in the action of this drug. Ó 2008 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Fasciola hepatica; Food-borne trematodiasis; 1,2,4 Trioxolanes; Synthetic peroxides; OZ78; In vivo studies; In vitro studies; Scanning electron microscopy
1. Introduction An estimated 91 million people are at risk and possibly as many as 17 million people are infected with Fasciola hepatica and F. gigantica (Keiser and Utzinger, 2005; WHO, 1995). Chronic infections with Fasciola spp. can cause severe disease manifestations, including hepatic lesions, fibrosis and chronic inflammation of the bile ducts (Richter et al., 2002). Fascioliasis is also of great veterinary importance causing considerable economic losses due to weight loss, reduction in milk yield and fertility in production animals (Schweizer et al., 2005). Chemotherapy is the current mainstay of control since there is no vaccine available for the prevention of fascioli*
Corresponding author. Fax: +41 61 284 8105. E-mail address: [email protected] (J. Keiser).
0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.12.015
asis (McManus and Dalton, 2006). Triclabendazole is the current drug of choice in human and veterinary medicine due to its high efficacy against both juvenile and adult F. hepatica and its excellent safety profile. In fascioliasis, in particular the immature parasite is highly pathogenic, causing fibrosis, hemorrhaging and anemia (Fairweather, 2005). Alternative drugs, which are effective against both parasite stages are presently not available and there is considerable concern of drug resistance development. Triclabendazole resistance has been first reported from sheep and cattle in Australia in the mid 1990s and has been spreading over Europe, the Netherlands, UK, Ireland or Spain (Alvarez-Sanchez et al., 2006; Keiser et al., 2005). There is an increasing awareness that novel trematocidal drugs should be identified. We have documented the promising fasciocidal properties of the antimalarial artemisinin derivatives artemether
88
J. Keiser, G. Morson / Experimental Parasitology 119 (2008) 87–93
and artesunate (Keiser et al., 2006a). A synthetic derivative, the 1,2,4 trioxolane OZ78, which possesses improved chemical and biopharmaceutical parameters when compared to the artemisinins (Vennerstrom et al., 2004) was also highly efficacious against adult and juvenile F. hepatica in rats; complete worm burden reductions were obtained at a single oral dose of 100 mg/kg (Keiser et al., 2006b). Importantly, OZ78 at 100 mg/kg cured rats harboring a triclabendazole resistant F. hepatica infection (Keiser et al., 2007a). The aim of this study was to assess tegumental changes in adult F. hepatica recovered from rats after administration of OZ78 by means of scanning electron microscopy (SEM). We furthermore document drug induced alterations on the surface morphology of F. hepatica following incubation with OZ78 in vitro, both in the absence and presence of haemin. 2. Materials and methods 2.1. Ethical clearance, parasites and host–parasite model All animal studies were carried out at the Swiss Tropical Institute (Basel, Switzerland) and had been approved by regulatory authorities following Swiss national regulations. Female Wistar rats (n = 12, age: 5 weeks, weight: 100 g) were purchased from RCC (Itingen, Switzerland). Rats were kept in groups of four in macrolon cages in environmentally-controlled conditions (temperature: 25 °C; humidity: 70%; 12 h light/dark cycle) and acclimatized for 1 week. They had free access to water and rodent diet. Metacercariae of F. hepatica (Cullompton isolate) were purchased from G. Graham (Addlestone, UK). 2.2. Drug The chemical structure of OZ78 is depicted in Fig. 1. OZ78 was synthesized at the College of Pharmacy, University of Nebraska Medical Center (Nebraska, USA). OZ78 was prepared in a suspension of 7% (v/v) Tween80 and 3% (v/v) ethanol before oral administration to rats. For the in vitro studies, a stock solution of OZ78 was prepared in 60% DMSO (v/v). 2.3. In vivo SEM observations on F. hepatica Rats, infected intragastrically with 25 metacercarial cysts of F. hepatica, were administered a single oral dose of 100 mg/kg OZ78 at week 12 post-infection. Two rats each were killed by CO2 at 24 and 48 h post-treatment. Four rats were examined 72 h post-treatment. At necropsy O O O
COOH
Fig. 1. Chemical structure of OZ78.
F. hepatica were recovered from the excised bile ducts and processed for SEM studies as described below. Flukes recovered from untreated rats served as controls. 2.4. In vitro studies Adult F. hepatica recovered from untreated rats were quickly rinsed in 0.9% (w/v) NaCl and incubated in 6-well plates (Costar), placing two F. hepatica per well. Culture medium in each well contained 5 ml NCTC 135 (Gibco), which was supplemented with antibiotics (50 lg/ml streptomycin and 50 U/ml penicillin; Gibco). For one series of experiments a haemin solution (80 lg/ml) was added to the medium: 5 mg of haemin was dissolved in 1 ml of 0.1 M aqueous solution of NaOH, and 3.95 ml of PBS (pH = 7.4) and 0.05 ml of 1 M HCl were added to adjust the pH to 7.2–7.4. The flukes were incubated with 10 lg/ ml OZ78 for 48 h. Controls were prepared by incubating flukes in 5 ml NCTC 135 supplemented with antibiotics, haemin and 0.06% DMSO for 48 h. Cultures were kept at 37 °C in an atmosphere of 5% CO2 and observed after exposure for 24 h and 48 h under a dissecting microscope. F. hepatica were collected after exposure to OZ78 for 48 h and processed for SEM observation as described below. 2.5. SEM observations Adult F. hepatica were rinsed with 0.9% NaCl (w/v) and fixed with 2.5% (v/v) glutaraldehyde in a PBS buffer for 24 h at room temperature. After rinsing with PBS buffer, the specimens were washed with distilled water, dehydrated with ethanol and critically point dried (Bomar SPC-900; Tacoma, USA). F. hepatica were mounted on aluminum stubs, sputter-coated with gold of 20 nm (Baltec Med 020, Tucson, USA) and observed in a high-resolution SEM (Philips XL30 ESEM; Eindhofen, the Netherlands) at an accelerating voltage of 5 kV. 3. Results 3.1. Control specimen We recovered between 4 and 7 flukes from untreated F. hepatica-infected rats. The surface of control specimen from in vivo and in vitro studies (with and without haemin) appeared normal as presented in a previous publication (Keiser and Morson, 2008). 3.2. In vivo SEM observations Six and three flukes, respectively, were recovered from the central bile ducts from two rats 24 h post-treatment with 100 mg/kg OZ78. All flukes were alive and showed normal movements. SEM observation revealed very minor damage. Only three F. hepatica showed slight impairment of the tegument. One F. hepatica revealed blebbing between the oral and the ventral sucker (Fig. 2a). Swelling below the
J. Keiser, G. Morson / Experimental Parasitology 119 (2008) 87–93
ventral sucker, resulting in a submerging of spines (Fig. 2b) and blebbing on the ventral mid-body (Fig. 2c) was observed on another specimen. Swelling on the dorsal tail surface (Fig. 2d) was visible on one of the flukes. The spines appeared slightly sunken due to the swelling. No tegumental alterations were observed on six flukes examined. Five F. hepatica recovered 2 days after oral administration with a single oral dose of 100 mg/kg OZ78 showed still normal activity. SEM observations revealed small damage of the tegument. However, alteration of the tegument was slightly more pronounced when compared to the 24 h time point. Overall, no clear dorsal ventral difference in tegument disruption was observed, but the posterior region was more affected than the anterior region of F. hepatica. All F. hepatica revealed blebbing on their ventral and dorsal mid-body surfaces, these areas of blebbing gave the tegument a roughened appearance (Fig. 3a–c). Swelling and extensive furrowing was visible on the dorsal and ventral (Fig. 3a and b) mid-body and tail surfaces of all F. hepatica examined. Often the spines were almost submerged by the surrounding swollen tegument (Fig. 3a and b). No tegumental alterations were observed in the oral cone region of both surfaces of F. hepatica examined. Three days post-treatment the recovery of F. hepatica from treated rats was low: all flukes had been expelled from the bile ducts of two rats. Incision of the swollen bile duct, revealed only yellow greenish liquid discharge and debris. F. hepatica from two further rats were either dead (n = 2) or showed only feeble activity and a pale appearance (n = 6). SEM analysis of the latter flukes showed extensive disruption of the ventral and dorsal tegument, particularly the tail region. Similar to the 48 h time point, no clear dor-
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sal ventral difference in tegument damage was observed, but the posterior region of the flukes was more affected than the anterior region. Disruption of the tegument of the flukes analyzed was considerably more severe than that observed after the 24 h and 48 h treatment time point. Again large areas of blebbing were observed, adding the tegument a roughened appearance (Fig. 4a). No damage was observed in the ventral and dorsal oral cone region, with the exception of the presence of a few blebs in the oral sucker of one specimen (Fig. 4b). Similar to the flukes sampled 48 h post-treatment tegumental damage as swelling, blebbing, sloughing, extensive furrowing, or eruptions was visible on the ventral (Fig. 4c) mid-body and dorsal (Fig. 4d) mid-body surfaces. Extensive sloughing was visible on the posterior mid-body and the tail region of all flukes examined (Fig. 4e–h). In the entire dorsal and ventral tail regions of the flukes the apical tegumental membrane had been removed. Large areas were completely devoid of spines, exposing spine sockets in the syncytium (Fig. 4e–h).
3.3. In vitro SEM observations 3.3.1. Medium without haemin Fasciola hepatica exposed for 24 h to 10 lg/ml OZ78 showed normal movements. After exposure for 48 h, the flukes showed reduced activity, extensive blebbing and several flukes had died. SEM analysis showed only minor disruption of the tegument. Two flukes examined revealed no tegumental damage. Blebs were present posteriad the ventral sucker (Fig. 5a) and on the ventral mid-body of one
Fig. 2. SEM of adult F. hepatica 24 h post-treatment with a single 100 mg/kg oral dose of OZ78. (a) Blebbing (b) between the oral sucker (OS) and the ventral sucker. (b) Swelling (sw) on the ventral mid-body tegument. (c) Blebbing (b) on the ventral mid-body. (d) Swelling (sw) on the dorsal tail region.
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Fig. 3. SEM of adult F. hepatica 48 h post-treatment with a single 100 mg/kg oral dose of OZ78. (a) Swelling (sw) and furrowing (f) on the ventral midbody. (b) Swelling (sw), blebbing (b) and furrowing (f) on the dorsal mid-body. (c) Blebbing (b) on the dorsal tail region of a F. hepatica.
fluke (Fig. 5b). Eruptions were seen on the ventral surface of four F. hepatica examined (Fig. 5c and d). 3.3.2. Medium supplemented with haemin Fasciola hepatica exposed to 10 lg/ml OZ78 in a medium supplemented with haemin were still alive after 24 h and were moving actively. Half of the flukes (4/8) died 48 h after incubation with OZ78 at this concentration. SEM pictures taken from flukes after exposure to 10 lg/ ml OZ78 in the presence of haemin revealed extensive damage of the tegument. All four flukes had massive damage of the anterior tegument. Large bands of blebs, furrows, and sloughing revealing empty spine sockets were observed near both suckers of all four flukes examined (Fig. 6a and b). No damage was observed on the ventral mid-body and tail of three F. hepatica incubated with OZ78 in the presence of haemin. One fluke showed a small area of sloughing on the ventral mid-body lateral margin (Fig. 6c). One F. hepatica showed blebbing and a large area of sloughing on the dorsal tail surface (Fig. 6d). 4. Discussion We have analyzed the effect of OZ78 on the tegumental surface of F. hepatica in vitro and in vivo. OZ78 is a synthetic 1,2,4 trioxolane, which is characterized by structural simplicity, ease of synthesis and improved pharmacokinetic properties when compared to the artemisinins (Vennerstrom et al., 2004). The present study has shown progressively severe disruption of the tegumental surface of F. hepatica confirming previous results that OZ78 displays promising fasciocidal activity (Keiser et al., 2006b). We
observed stress reactions such as blebbing and swelling of the tegument of F. hepatica 24 h post-treatment of rats with OZ78. This early response of F. hepatica to OZ78 lead on to extensive tegumental alterations such as sloughing 72 h post-treatment. Tegumental disruption was also observed after exposure of flukes to OZ78 in vitro in particular in the presence of haemin, suggesting that the tegument of F. hepatica might be a main drug target of OZ78. Morphological alterations such as sloughing, blebbing or swelling of the tegument have also been observed in a recent study, which analyzed the effect of OZ78 on the tegument of another major trematode, namely Clonorchis sinensis (Keiser et al., 2007b). Interestingly, extensive disruption of the tegumental surface of C. sinensis was already present 24 h post-treatment of rats, which did not deteriorate much further within the next 48 h. Specimens were still alive and moved actively 72 h post-treatment (Keiser et al., 2007b). Several findings of our study are worth highlighting. First, our in vitro experiments demonstrated that damage of the tegument of F. hepatica following incubation with OZ78 in vitro was more pronounced when haemin was added to the incubation medium. Interestingly, however parasite death was not accelerated through haemin supplementation. We have, not surprisingly, observed the same finding in a recent study analyzing the effect of artemether and artesunate on the tegument of F. hepatica. When haemin was added to the incubation medium containing artesunate or artemether, F. hepatica showed pronounced tegumental alterations (Keiser and Morson, 2008). However, parasite death was also not influenced by haemin supplementation. We have speculated that the formation of
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Fig. 4. SEM of adult F. hepatica 72 h post-treatment with a single 100 mg/kg oral dose of OZ78. (a) Large areas of blebbing (b) on the dorsal surface of a fluke (OS = oral sucker). (b) Blebs (b) present in the oral sucker. (c) Blebbing (b) and swelling (sw) on the ventral mid-body surface. (d) Swelling (sw), sloughing (s), empty spine sockets (SS) and eruption (e) on the dorsal mid-body tegument. (e) Sloughing (s), empty spine sockets (SS) blebbing (b) on the dorsal tail region. (f–h) Empty spine sockets (SS) on the ventral tail surface.
free radicals mediated by iron, which possibly react with different targets as different proteins or proteinases (one of the mechanisms of action attributed to the antiplasmodial action of the artemisinins (Meshnick, 2002)) might also play a role but might not be the sole mechanism of action involved in the death of F. hepatica (Keiser and Morson, 2008). Similarly, haemin might play a role in the mechanism of action of OZ78 on F. hepatica, but other drug induced processes leading to parasite death might also be involved.
Second, tegumental alterations following in vitro incubation with OZ78, both in the presence and absence of haemin differed from changes on the tegument observed on F. hepatica recovered from rats, which were treated with OZ78. While in vivo specimens showed extensive damage of the posterior region, in vitro specimens showed either eruptions on the mid-body (no haemin added) or mostly substantial damage of the anterior part, including the suckers of F. hepatica (haemin supplemented). Regional variations of the drug effect
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Fig. 5. SEM of adult F. hepatica 48 h post-incubation in vitro with 10 lg/ml OZ78. (a) Area of blebs (b) below the ventral sucker (VS). (b) blebbing (b) on the ventral mid-body. (c and d) Eruption (e) on the ventral mid-body.
Fig. 6. SEM of adult F. hepatica 48 h post-incubation in vitro with 10 lg/ml OZ78 in the presence of haemin. (a) Sloughing (s), furrowing (f) and empty spine sockets (SS) visible near the oral sucker (OS). (b) Blebbing (b) and sloughing (s) and empty spine sockets (SS) near the ventral sucker (VS). (c) Blebbing (b) and sloughing (s) on the ventral mid-body lateral margin. (d) Blebbing (b) and sloughing (s) on the dorsal tail region.
between in vitro and in vivo specimens might be explained by the presence of active metabolites, protein binding or different feeding ability in vivo. Further studies, aiming to elucidate biopharmaceutical and pharmacokinetic properties including metabolism of OZ78 are ongoing in our laboratories, which might help to answer these questions.
Third, differences were observed between two peroxidic drugs, namely OZ78 and artemether in respect to progression of the tegumental surface alterations. While extensive damage was detected on F. hepatica collected from rats already 24 h post-treatment with artemether, flukes treated with OZ78 showed still relatively minor damage 48 h posttreatment. Interestingly, there is quite a time lag between
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the short half live of the OZ78, i.e. 2 h in rats, and the onset of the most severe disruption of the tegument observed at 72 h, which cannot be explained at the moment. Damage of the tegument exposes sub-tegument tissues to be attacked by the bile, which might result in greater tegumental damage or even death of flukes. Damage to internal organs following drug penetration might also be involved in the drug action of OZ78 on Fasciola spp. These changes to the internal tissues remain to be investigated, and TEM observations using a similar study design lend themselves to accomplish this objective. Acknowledgments J. Keiser (Project No. PMPDB-114358) is grateful to the Swiss National Science Foundation for financial support. We thank Jonathan Vennerstrom for supplying us with OZ78 and his continuous support. References Alvarez-Sanchez, M.A., Mainar-Jaime, R.C., Perez-Garcia, J., RojoVazquez, F.A., 2006. Resistance of Fasciola hepatica to triclabendazole and albendazole in sheep in Spain. Veterinary Records 159, 424–425. Fairweather, I., 2005. Triclabendazole: new skills to unravel an old(ish) enigma. Journal of Helminthology 79, 227–234. Keiser, J., Engels, D., Bu¨scher, G., Utzinger, J., 2005. Triclabendazole for the treatment of fascioliasis and paragonimiasis. Expert Opinion Investigational Drugs 14, 1513–1526. Keiser, J., Morson, G., 2008. Fasciola hepatica: tegumental alterations in adult flukes following in vitro and in vivo administration of artesunate and artemether. Experimental Parasitology 118, 228–237.
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Keiser, J., Xiao, S.H., Tanner, M., Utzinger, J., 2006a. Artesunate and artemether are effective fasciolicides in the rat model and in vitro. Journal of Antimicrobial Chemotherapy 57, 1139–1145. Keiser, J., Utzinger, J., 2005. Emerging foodborne trematodiasis. Emerging Infectious Diseases 11, 1507–1514. Keiser, J., Utzinger, J., Tanner, M., Dong, Y., Vennerstrom, J.L., 2006b. The synthetic peroxide OZ78 is effective against Echinostoma caproni and Fasciola hepatica. Journal of Antimicrobial Chemotherapy 58, 1193–1197. Keiser, J., Utzinger, J., Vennerstrom, J.L., Dong, Y., Brennan, G.P., Fairweather, I., 2007a. Activity of artemether and OZ78 against triclabendazole-resistant Fasciola hepatica. Transactions of the Royal Society of Tropical Medicine and Hygiene 101, 1219–1222. Keiser, J., Xiao, S.H., Dong, Y., Utzinger, J., Vennerstrom, J.L., 2007b. Clonorchicidal properties of the synthetic trioxolane OZ78. Journal of Parasitology 93, 1208–1213. McManus, D.P., Dalton, J.P., 2006. Vaccines against the zoonotic trematodes Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica. Parasitology 133 (Suppl.), S43–S61. Meshnick, S.R., 2002. Artemisinin: mechanisms of action, resistance and toxicity. International Journal for Parasitology 32, 1655–1660. Richter, J., Knipper, M., Goebels, K., Haeussinger, D., 2002. Fascioliasis. Current Treatment Options in Infectious Diseases 4, 313–317. Schweizer, G., Braun, U., Deplazes, P., Torgerson, P.R., 2005. Estimating the financial losses due to bovine fasciolosis in Switzerland. Veterinary Records 157, 188–193. Vennerstrom, J.L., Arbe-Barnes, S., Brun, R., Charman, S.A., Chiu, F.C., Chollet, J., Dong, Y., Dorn, A., Hunziker, D., Matile, H., McIntosh, K., Padmanilayam, M., Santo Tomas, J., Scheurer, C., Scorneaux, B., Tang, Y., Urwyler, H., Wittlin, S., Charman, W.N., 2004. Identification of an antimalarial synthetic trioxolane drug development candidate. Nature 430, 900–904. WHO, 1995. Control of foodborne trematode infections. Report of a WHO study group. WHO Tech. Rep. Ser. No. 849. World Health Organization, Geneva.
Experimental Parasitology 119 (2008) 87–93 www.elsevier.com/locate/yexpr
Fasciola hepatica: Surface tegumental responses to in vitro and in vivo treatment with the experimental fasciolicide OZ78 Jennifer Keiser a,*, Gianni Morson b b
a Swiss Tropical Institute, Medical Parasitology and Infection Biology, Socinstr. 57, P.O. Box, CH–4002 Basel, Switzerland Center for Microscopy, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, University of Basel, Switzerland
Received 30 October 2007; received in revised form 17 December 2007; accepted 30 December 2007 Available online 8 January 2008
Abstract The tegumental alterations in adult Fasciola hepatica induced by the experimental fasciolide OZ78 were investigated utilizing scanning electron microscopy (SEM). Twelve weeks post-infection with F. hepatica, rats were treated with a single 100 mg/kg oral dose of OZ78 and flukes were recovered from the bile ducts after 24–72 h. In vitro F. hepatica were incubated with OZ78 for 48 h at a concentration of 10 lg/ml in the absence or presence of haemin. Twenty-four and 48 h post-treatment of rats disruption of the tegument of F. hepatica as blebbing, swelling and furrowing was evident. The recovery of flukes 72 h post-treatment was low. Flukes examined at this time point showed an increasing severity of tegumental damage as sloughing and absence of spines, in particular in the tail region. SEM analysis of F. hepatica incubated in the presence of OZ78 without haemin showed only minor and localized damage of the tegument. In the presence of haemin extensive tegumental damage, including sloughing or blebbing, in particular in the anterior part, was observed. In conclusion, our experiments confirm the interesting fasciocidal properties of OZ78. The tegument of adult F. hepatica might play a role in the action of this drug. Ó 2008 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Fasciola hepatica; Food-borne trematodiasis; 1,2,4 Trioxolanes; Synthetic peroxides; OZ78; In vivo studies; In vitro studies; Scanning electron microscopy
1. Introduction An estimated 91 million people are at risk and possibly as many as 17 million people are infected with Fasciola hepatica and F. gigantica (Keiser and Utzinger, 2005; WHO, 1995). Chronic infections with Fasciola spp. can cause severe disease manifestations, including hepatic lesions, fibrosis and chronic inflammation of the bile ducts (Richter et al., 2002). Fascioliasis is also of great veterinary importance causing considerable economic losses due to weight loss, reduction in milk yield and fertility in production animals (Schweizer et al., 2005). Chemotherapy is the current mainstay of control since there is no vaccine available for the prevention of fascioli*
Corresponding author. Fax: +41 61 284 8105. E-mail address: [email protected] (J. Keiser).
0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.12.015
asis (McManus and Dalton, 2006). Triclabendazole is the current drug of choice in human and veterinary medicine due to its high efficacy against both juvenile and adult F. hepatica and its excellent safety profile. In fascioliasis, in particular the immature parasite is highly pathogenic, causing fibrosis, hemorrhaging and anemia (Fairweather, 2005). Alternative drugs, which are effective against both parasite stages are presently not available and there is considerable concern of drug resistance development. Triclabendazole resistance has been first reported from sheep and cattle in Australia in the mid 1990s and has been spreading over Europe, the Netherlands, UK, Ireland or Spain (Alvarez-Sanchez et al., 2006; Keiser et al., 2005). There is an increasing awareness that novel trematocidal drugs should be identified. We have documented the promising fasciocidal properties of the antimalarial artemisinin derivatives artemether
88
J. Keiser, G. Morson / Experimental Parasitology 119 (2008) 87–93
and artesunate (Keiser et al., 2006a). A synthetic derivative, the 1,2,4 trioxolane OZ78, which possesses improved chemical and biopharmaceutical parameters when compared to the artemisinins (Vennerstrom et al., 2004) was also highly efficacious against adult and juvenile F. hepatica in rats; complete worm burden reductions were obtained at a single oral dose of 100 mg/kg (Keiser et al., 2006b). Importantly, OZ78 at 100 mg/kg cured rats harboring a triclabendazole resistant F. hepatica infection (Keiser et al., 2007a). The aim of this study was to assess tegumental changes in adult F. hepatica recovered from rats after administration of OZ78 by means of scanning electron microscopy (SEM). We furthermore document drug induced alterations on the surface morphology of F. hepatica following incubation with OZ78 in vitro, both in the absence and presence of haemin. 2. Materials and methods 2.1. Ethical clearance, parasites and host–parasite model All animal studies were carried out at the Swiss Tropical Institute (Basel, Switzerland) and had been approved by regulatory authorities following Swiss national regulations. Female Wistar rats (n = 12, age: 5 weeks, weight: 100 g) were purchased from RCC (Itingen, Switzerland). Rats were kept in groups of four in macrolon cages in environmentally-controlled conditions (temperature: 25 °C; humidity: 70%; 12 h light/dark cycle) and acclimatized for 1 week. They had free access to water and rodent diet. Metacercariae of F. hepatica (Cullompton isolate) were purchased from G. Graham (Addlestone, UK). 2.2. Drug The chemical structure of OZ78 is depicted in Fig. 1. OZ78 was synthesized at the College of Pharmacy, University of Nebraska Medical Center (Nebraska, USA). OZ78 was prepared in a suspension of 7% (v/v) Tween80 and 3% (v/v) ethanol before oral administration to rats. For the in vitro studies, a stock solution of OZ78 was prepared in 60% DMSO (v/v). 2.3. In vivo SEM observations on F. hepatica Rats, infected intragastrically with 25 metacercarial cysts of F. hepatica, were administered a single oral dose of 100 mg/kg OZ78 at week 12 post-infection. Two rats each were killed by CO2 at 24 and 48 h post-treatment. Four rats were examined 72 h post-treatment. At necropsy O O O
COOH
Fig. 1. Chemical structure of OZ78.
F. hepatica were recovered from the excised bile ducts and processed for SEM studies as described below. Flukes recovered from untreated rats served as controls. 2.4. In vitro studies Adult F. hepatica recovered from untreated rats were quickly rinsed in 0.9% (w/v) NaCl and incubated in 6-well plates (Costar), placing two F. hepatica per well. Culture medium in each well contained 5 ml NCTC 135 (Gibco), which was supplemented with antibiotics (50 lg/ml streptomycin and 50 U/ml penicillin; Gibco). For one series of experiments a haemin solution (80 lg/ml) was added to the medium: 5 mg of haemin was dissolved in 1 ml of 0.1 M aqueous solution of NaOH, and 3.95 ml of PBS (pH = 7.4) and 0.05 ml of 1 M HCl were added to adjust the pH to 7.2–7.4. The flukes were incubated with 10 lg/ ml OZ78 for 48 h. Controls were prepared by incubating flukes in 5 ml NCTC 135 supplemented with antibiotics, haemin and 0.06% DMSO for 48 h. Cultures were kept at 37 °C in an atmosphere of 5% CO2 and observed after exposure for 24 h and 48 h under a dissecting microscope. F. hepatica were collected after exposure to OZ78 for 48 h and processed for SEM observation as described below. 2.5. SEM observations Adult F. hepatica were rinsed with 0.9% NaCl (w/v) and fixed with 2.5% (v/v) glutaraldehyde in a PBS buffer for 24 h at room temperature. After rinsing with PBS buffer, the specimens were washed with distilled water, dehydrated with ethanol and critically point dried (Bomar SPC-900; Tacoma, USA). F. hepatica were mounted on aluminum stubs, sputter-coated with gold of 20 nm (Baltec Med 020, Tucson, USA) and observed in a high-resolution SEM (Philips XL30 ESEM; Eindhofen, the Netherlands) at an accelerating voltage of 5 kV. 3. Results 3.1. Control specimen We recovered between 4 and 7 flukes from untreated F. hepatica-infected rats. The surface of control specimen from in vivo and in vitro studies (with and without haemin) appeared normal as presented in a previous publication (Keiser and Morson, 2008). 3.2. In vivo SEM observations Six and three flukes, respectively, were recovered from the central bile ducts from two rats 24 h post-treatment with 100 mg/kg OZ78. All flukes were alive and showed normal movements. SEM observation revealed very minor damage. Only three F. hepatica showed slight impairment of the tegument. One F. hepatica revealed blebbing between the oral and the ventral sucker (Fig. 2a). Swelling below the
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ventral sucker, resulting in a submerging of spines (Fig. 2b) and blebbing on the ventral mid-body (Fig. 2c) was observed on another specimen. Swelling on the dorsal tail surface (Fig. 2d) was visible on one of the flukes. The spines appeared slightly sunken due to the swelling. No tegumental alterations were observed on six flukes examined. Five F. hepatica recovered 2 days after oral administration with a single oral dose of 100 mg/kg OZ78 showed still normal activity. SEM observations revealed small damage of the tegument. However, alteration of the tegument was slightly more pronounced when compared to the 24 h time point. Overall, no clear dorsal ventral difference in tegument disruption was observed, but the posterior region was more affected than the anterior region of F. hepatica. All F. hepatica revealed blebbing on their ventral and dorsal mid-body surfaces, these areas of blebbing gave the tegument a roughened appearance (Fig. 3a–c). Swelling and extensive furrowing was visible on the dorsal and ventral (Fig. 3a and b) mid-body and tail surfaces of all F. hepatica examined. Often the spines were almost submerged by the surrounding swollen tegument (Fig. 3a and b). No tegumental alterations were observed in the oral cone region of both surfaces of F. hepatica examined. Three days post-treatment the recovery of F. hepatica from treated rats was low: all flukes had been expelled from the bile ducts of two rats. Incision of the swollen bile duct, revealed only yellow greenish liquid discharge and debris. F. hepatica from two further rats were either dead (n = 2) or showed only feeble activity and a pale appearance (n = 6). SEM analysis of the latter flukes showed extensive disruption of the ventral and dorsal tegument, particularly the tail region. Similar to the 48 h time point, no clear dor-
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sal ventral difference in tegument damage was observed, but the posterior region of the flukes was more affected than the anterior region. Disruption of the tegument of the flukes analyzed was considerably more severe than that observed after the 24 h and 48 h treatment time point. Again large areas of blebbing were observed, adding the tegument a roughened appearance (Fig. 4a). No damage was observed in the ventral and dorsal oral cone region, with the exception of the presence of a few blebs in the oral sucker of one specimen (Fig. 4b). Similar to the flukes sampled 48 h post-treatment tegumental damage as swelling, blebbing, sloughing, extensive furrowing, or eruptions was visible on the ventral (Fig. 4c) mid-body and dorsal (Fig. 4d) mid-body surfaces. Extensive sloughing was visible on the posterior mid-body and the tail region of all flukes examined (Fig. 4e–h). In the entire dorsal and ventral tail regions of the flukes the apical tegumental membrane had been removed. Large areas were completely devoid of spines, exposing spine sockets in the syncytium (Fig. 4e–h).
3.3. In vitro SEM observations 3.3.1. Medium without haemin Fasciola hepatica exposed for 24 h to 10 lg/ml OZ78 showed normal movements. After exposure for 48 h, the flukes showed reduced activity, extensive blebbing and several flukes had died. SEM analysis showed only minor disruption of the tegument. Two flukes examined revealed no tegumental damage. Blebs were present posteriad the ventral sucker (Fig. 5a) and on the ventral mid-body of one
Fig. 2. SEM of adult F. hepatica 24 h post-treatment with a single 100 mg/kg oral dose of OZ78. (a) Blebbing (b) between the oral sucker (OS) and the ventral sucker. (b) Swelling (sw) on the ventral mid-body tegument. (c) Blebbing (b) on the ventral mid-body. (d) Swelling (sw) on the dorsal tail region.
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Fig. 3. SEM of adult F. hepatica 48 h post-treatment with a single 100 mg/kg oral dose of OZ78. (a) Swelling (sw) and furrowing (f) on the ventral midbody. (b) Swelling (sw), blebbing (b) and furrowing (f) on the dorsal mid-body. (c) Blebbing (b) on the dorsal tail region of a F. hepatica.
fluke (Fig. 5b). Eruptions were seen on the ventral surface of four F. hepatica examined (Fig. 5c and d). 3.3.2. Medium supplemented with haemin Fasciola hepatica exposed to 10 lg/ml OZ78 in a medium supplemented with haemin were still alive after 24 h and were moving actively. Half of the flukes (4/8) died 48 h after incubation with OZ78 at this concentration. SEM pictures taken from flukes after exposure to 10 lg/ ml OZ78 in the presence of haemin revealed extensive damage of the tegument. All four flukes had massive damage of the anterior tegument. Large bands of blebs, furrows, and sloughing revealing empty spine sockets were observed near both suckers of all four flukes examined (Fig. 6a and b). No damage was observed on the ventral mid-body and tail of three F. hepatica incubated with OZ78 in the presence of haemin. One fluke showed a small area of sloughing on the ventral mid-body lateral margin (Fig. 6c). One F. hepatica showed blebbing and a large area of sloughing on the dorsal tail surface (Fig. 6d). 4. Discussion We have analyzed the effect of OZ78 on the tegumental surface of F. hepatica in vitro and in vivo. OZ78 is a synthetic 1,2,4 trioxolane, which is characterized by structural simplicity, ease of synthesis and improved pharmacokinetic properties when compared to the artemisinins (Vennerstrom et al., 2004). The present study has shown progressively severe disruption of the tegumental surface of F. hepatica confirming previous results that OZ78 displays promising fasciocidal activity (Keiser et al., 2006b). We
observed stress reactions such as blebbing and swelling of the tegument of F. hepatica 24 h post-treatment of rats with OZ78. This early response of F. hepatica to OZ78 lead on to extensive tegumental alterations such as sloughing 72 h post-treatment. Tegumental disruption was also observed after exposure of flukes to OZ78 in vitro in particular in the presence of haemin, suggesting that the tegument of F. hepatica might be a main drug target of OZ78. Morphological alterations such as sloughing, blebbing or swelling of the tegument have also been observed in a recent study, which analyzed the effect of OZ78 on the tegument of another major trematode, namely Clonorchis sinensis (Keiser et al., 2007b). Interestingly, extensive disruption of the tegumental surface of C. sinensis was already present 24 h post-treatment of rats, which did not deteriorate much further within the next 48 h. Specimens were still alive and moved actively 72 h post-treatment (Keiser et al., 2007b). Several findings of our study are worth highlighting. First, our in vitro experiments demonstrated that damage of the tegument of F. hepatica following incubation with OZ78 in vitro was more pronounced when haemin was added to the incubation medium. Interestingly, however parasite death was not accelerated through haemin supplementation. We have, not surprisingly, observed the same finding in a recent study analyzing the effect of artemether and artesunate on the tegument of F. hepatica. When haemin was added to the incubation medium containing artesunate or artemether, F. hepatica showed pronounced tegumental alterations (Keiser and Morson, 2008). However, parasite death was also not influenced by haemin supplementation. We have speculated that the formation of
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Fig. 4. SEM of adult F. hepatica 72 h post-treatment with a single 100 mg/kg oral dose of OZ78. (a) Large areas of blebbing (b) on the dorsal surface of a fluke (OS = oral sucker). (b) Blebs (b) present in the oral sucker. (c) Blebbing (b) and swelling (sw) on the ventral mid-body surface. (d) Swelling (sw), sloughing (s), empty spine sockets (SS) and eruption (e) on the dorsal mid-body tegument. (e) Sloughing (s), empty spine sockets (SS) blebbing (b) on the dorsal tail region. (f–h) Empty spine sockets (SS) on the ventral tail surface.
free radicals mediated by iron, which possibly react with different targets as different proteins or proteinases (one of the mechanisms of action attributed to the antiplasmodial action of the artemisinins (Meshnick, 2002)) might also play a role but might not be the sole mechanism of action involved in the death of F. hepatica (Keiser and Morson, 2008). Similarly, haemin might play a role in the mechanism of action of OZ78 on F. hepatica, but other drug induced processes leading to parasite death might also be involved.
Second, tegumental alterations following in vitro incubation with OZ78, both in the presence and absence of haemin differed from changes on the tegument observed on F. hepatica recovered from rats, which were treated with OZ78. While in vivo specimens showed extensive damage of the posterior region, in vitro specimens showed either eruptions on the mid-body (no haemin added) or mostly substantial damage of the anterior part, including the suckers of F. hepatica (haemin supplemented). Regional variations of the drug effect
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Fig. 5. SEM of adult F. hepatica 48 h post-incubation in vitro with 10 lg/ml OZ78. (a) Area of blebs (b) below the ventral sucker (VS). (b) blebbing (b) on the ventral mid-body. (c and d) Eruption (e) on the ventral mid-body.
Fig. 6. SEM of adult F. hepatica 48 h post-incubation in vitro with 10 lg/ml OZ78 in the presence of haemin. (a) Sloughing (s), furrowing (f) and empty spine sockets (SS) visible near the oral sucker (OS). (b) Blebbing (b) and sloughing (s) and empty spine sockets (SS) near the ventral sucker (VS). (c) Blebbing (b) and sloughing (s) on the ventral mid-body lateral margin. (d) Blebbing (b) and sloughing (s) on the dorsal tail region.
between in vitro and in vivo specimens might be explained by the presence of active metabolites, protein binding or different feeding ability in vivo. Further studies, aiming to elucidate biopharmaceutical and pharmacokinetic properties including metabolism of OZ78 are ongoing in our laboratories, which might help to answer these questions.
Third, differences were observed between two peroxidic drugs, namely OZ78 and artemether in respect to progression of the tegumental surface alterations. While extensive damage was detected on F. hepatica collected from rats already 24 h post-treatment with artemether, flukes treated with OZ78 showed still relatively minor damage 48 h posttreatment. Interestingly, there is quite a time lag between
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the short half live of the OZ78, i.e. 2 h in rats, and the onset of the most severe disruption of the tegument observed at 72 h, which cannot be explained at the moment. Damage of the tegument exposes sub-tegument tissues to be attacked by the bile, which might result in greater tegumental damage or even death of flukes. Damage to internal organs following drug penetration might also be involved in the drug action of OZ78 on Fasciola spp. These changes to the internal tissues remain to be investigated, and TEM observations using a similar study design lend themselves to accomplish this objective. Acknowledgments J. Keiser (Project No. PMPDB-114358) is grateful to the Swiss National Science Foundation for financial support. We thank Jonathan Vennerstrom for supplying us with OZ78 and his continuous support. References Alvarez-Sanchez, M.A., Mainar-Jaime, R.C., Perez-Garcia, J., RojoVazquez, F.A., 2006. Resistance of Fasciola hepatica to triclabendazole and albendazole in sheep in Spain. Veterinary Records 159, 424–425. Fairweather, I., 2005. Triclabendazole: new skills to unravel an old(ish) enigma. Journal of Helminthology 79, 227–234. Keiser, J., Engels, D., Bu¨scher, G., Utzinger, J., 2005. Triclabendazole for the treatment of fascioliasis and paragonimiasis. Expert Opinion Investigational Drugs 14, 1513–1526. Keiser, J., Morson, G., 2008. Fasciola hepatica: tegumental alterations in adult flukes following in vitro and in vivo administration of artesunate and artemether. Experimental Parasitology 118, 228–237.
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Keiser, J., Xiao, S.H., Tanner, M., Utzinger, J., 2006a. Artesunate and artemether are effective fasciolicides in the rat model and in vitro. Journal of Antimicrobial Chemotherapy 57, 1139–1145. Keiser, J., Utzinger, J., 2005. Emerging foodborne trematodiasis. Emerging Infectious Diseases 11, 1507–1514. Keiser, J., Utzinger, J., Tanner, M., Dong, Y., Vennerstrom, J.L., 2006b. The synthetic peroxide OZ78 is effective against Echinostoma caproni and Fasciola hepatica. Journal of Antimicrobial Chemotherapy 58, 1193–1197. Keiser, J., Utzinger, J., Vennerstrom, J.L., Dong, Y., Brennan, G.P., Fairweather, I., 2007a. Activity of artemether and OZ78 against triclabendazole-resistant Fasciola hepatica. Transactions of the Royal Society of Tropical Medicine and Hygiene 101, 1219–1222. Keiser, J., Xiao, S.H., Dong, Y., Utzinger, J., Vennerstrom, J.L., 2007b. Clonorchicidal properties of the synthetic trioxolane OZ78. Journal of Parasitology 93, 1208–1213. McManus, D.P., Dalton, J.P., 2006. Vaccines against the zoonotic trematodes Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica. Parasitology 133 (Suppl.), S43–S61. Meshnick, S.R., 2002. Artemisinin: mechanisms of action, resistance and toxicity. International Journal for Parasitology 32, 1655–1660. Richter, J., Knipper, M., Goebels, K., Haeussinger, D., 2002. Fascioliasis. Current Treatment Options in Infectious Diseases 4, 313–317. Schweizer, G., Braun, U., Deplazes, P., Torgerson, P.R., 2005. Estimating the financial losses due to bovine fasciolosis in Switzerland. Veterinary Records 157, 188–193. Vennerstrom, J.L., Arbe-Barnes, S., Brun, R., Charman, S.A., Chiu, F.C., Chollet, J., Dong, Y., Dorn, A., Hunziker, D., Matile, H., McIntosh, K., Padmanilayam, M., Santo Tomas, J., Scheurer, C., Scorneaux, B., Tang, Y., Urwyler, H., Wittlin, S., Charman, W.N., 2004. Identification of an antimalarial synthetic trioxolane drug development candidate. Nature 430, 900–904. WHO, 1995. Control of foodborne trematode infections. Report of a WHO study group. WHO Tech. Rep. Ser. No. 849. World Health Organization, Geneva.