Veterinary Parasitology 201 (2014) 48–58

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The anthelmintic efficacy of natural plant cysteine proteinases against two rodent cestodes Hymenolepis diminuta and Hymenolepis microstoma in vitro F. Mansur a,c , W. Luoga a,d , D.J. Buttle b , I.R. Duce a , Ann Lowe a , J.M. Behnke a,∗ a b c d

School of Life Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK Department of Infection and Immunity, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK Faculty of Medicine and Health Sciences, Universiti Sains Islam Malaysia (USIM), Kuala Lumpur, Malaysia Department of Life Sciences, Mkwawa University College of Education, Iringa, Tanzania

a r t i c l e

i n f o

Article history: Received 19 September 2013 Received in revised form 18 December 2013 Accepted 19 December 2013

Keywords: Cysteine proteinases Anthelmintics Hymenolepis diminuta Hymenolepis microstoma Papaya latex

a b s t r a c t Little is known about the efficacy of cysteine proteinases (CP) as anthelmintics for cestode infections. We examined the effects of CPs on two rodent cestodes, Hymenolepis diminuta and H. microstoma in vitro. Our data showed that naturally occurring mixtures of CPs, such as those found in papaya latex, and relatively pure preparations of fruit bromelain, papain and stem bromelain, were active in vitro against both juvenile, artificially excysted scoleces, as well as against adult worms of both rodent cestodes. Significant dose-dependent reduction in motility, ultimately leading to death of the worms, was observed with both species, and against both freshly excysted scoleces and 14-day old pre-adult worms. The most effective was fruit bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50 = 63 and 74 ␮M, respectively, and for pre-adult worms = 199 and 260 ␮M, respectively). The least effective was stem bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50 = 2855 and 2772 ␮M, respectively, and for pre-adult worms = 1374 and 1332 ␮M, respectively) and the efficacies of papaya latex supernatant and papain were between these extremes. In all cases these values are higher than those reported previously for efficacy of CPs against intestinal nematodes, and in contrast to nematodes, all CPs were effective against cestodes in the absence of exogenous cysteine in incubation media. The CPs appeared to attack the tegument resulting in generalised erosion mainly on the strobila. The scolex was more resistant to CP attack but nevertheless some damage to the tegument on the scolex was detected. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.

1. Introduction Helminths are known to have afflicted humans and livestock since antiquity (Cox, 2004), and therefore it is no surprise to find that in the past traditional remedies, mostly

∗ Corresponding author at: School of Biology, University of Nottingham, University Park, Nottingham NG7 2RD, UK. Tel.: +44 115 951 3208; fax: +44 115 951 3251. E-mail address: [email protected] (J.M. Behnke).

from plant extracts have been used to treat them (Behnke et al., 2008). While ethno-medical evidence for the use of plants as anthelmintics abounds (Prakash and Mehrotra, 1987), historical records of the specific use of plant based cestocidals remain elusive, apart from the report of the use of powdered fern extract by ancient Greeks (350–250 B.C.) referred to in Nordic literature (Waller et al., 2001). There are many more recent reports in the literature of plants showing cestocidal activity, but with as yet unknown active principles, as for example in the case of Strobilates discolour (Tangpu et al., 2006), Cassia spp. (Kundu and Lyndem, 2012;

0304-4017/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.12.018

F. Mansur et al. / Veterinary Parasitology 201 (2014) 48–58

Kundu et al., 2012), Solenostemon spp. leaves and Croton spp. twigs (He et al., 1992). Scientific evidence of active principles mediating the cestocidal properties of plants is still limited and mostly not conclusively proven, but nevertheless the identities of the active principles from some plants reported to have cestocidal activity have been proposed. For example the tuberous root of the plant Flemingia vestita has been reported to contain an active principle thought to be genistein (Tandon et al., 2003), the alkaloid components of Adhatoda vasica are cestocidal (Yadav and Tangpu, 2008) and Acacia spp. contain saponins and tannins with cestocidal properties (Ghosh et al., 1996). However, despite the exciting prospect that some of these plant sources may eventually yield new cestocidal drugs, so far none have been tested rigorously and in no case have the active principles been proven conclusively to mediate the purported cestocidal activity. Plant extracts from pineapples, papaya and figs contain cysteine proteinases (CPs) which have been systematically tested against intestinal nematode parasites and their anthelmintic properties have been confirmed to be mediated by the CPs (Stepek et al., 2005). Therefore, CPs hold a real potential for development as mainstream anthelmintic drugs. However, in comparison to nematodes, little is still known about the effects of CPs on cestode infections. Evidence that CPs are detrimental to the survival of cestodes in vitro has been provided (He et al., 1992; Stepek et al., 2007c), but current data also indicate that this is not reflected in a reduction of parasite burdens when CPs are administered to infected hosts (de Amorin et al., 1999; He et al., 1992). In this paper we demonstrate that plant derived CPs are detrimental to cestodes maintained in temporary in vitro assays. We used two species of laboratory maintained cestodes, the murine species Hymenolepis microstoma and the rat parasite H. diminuta, and in both cases we assessed the effects of several CPs, at a range of concentrations, on newly excysted scoleces obtained from cysticercoids derived from beetles and on 14-day pre-adult worms extracted from infected rodent hosts.

2. Materials and methods 2.1. Enzyme preparation The enzymes used in this study were CPs derived from papaya latex supernatant (PLS) containing chymopapain, glycyl endopeptidase, caricain and papain (in order of relative abundance (Buttle et al., 1990)), pineapple fruit bromelain (FB), pineapple stem bromelain (SB) and papain. PLS was prepared as previously described by Buttle et al. (2011) and aliquoted into individual vials and stored at −80 ◦ C. The molar concentration of the active CP was measured by active site titration adapted from Barrett et al. (1981) and Zucker et al. (1985) with increasing concentrations of the CP specific inhibitor, ltrans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane (E-64) (Apollo Scientific Ltd., UK) with 4 mM l-cysteine as a reducing agent and benzoyl-arginyl-p-nitroanilide (BAPNA) (Bachem Ltd., UK) as the substrate.

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For preparation of FB we adapted the method of Rowan et al. (1990). Ripe fruits were purchased from Sainsbury’s supermarket, U.K., peeled, cut into small pieces and then crushed in a laboratory juicer (Philips juicer HR 1861) and filtered. The filtrate was then subjected to vacuum filtration (Whatman® quantitative filter paper, hardened ashless, Grade 540 diameter 110) to remove the remaining fine debris. The filtered juice was concentrated by dialysis over polyethylene glycol and the concentrated juice was aliquoted into vials and stored at −80 ◦ C for future use. The active enzyme concentrations from each stage of preparation were determined by active-site titration with E-64 using benzyloxycarbonyl-Phe-Arg-pNA (Z-Phe-ArgpNA) (Bachem Ltd., UK) as the substrate. Pineapple SB was obtained from Hong Mao Biochemical Co. Ltd, Thailand, kindly provided as a gift. Two hundred grams of SB powder were dissolved in 1 litre of distilled water and centrifuged at 17,700 × g at 4 ◦ C and the pellets were discarded while the supernatant was aliquoted into vials and stored at −80 ◦ C. Its enzyme activity was assessed by active site titration with E-64 using benzyloxycarbonylArg-Arg-pNA (Z-Arg-Arg-pNA) (Bachem Ltd., UK) as its substrate. On the day of each experiment, individual vials were thawed and assessed for active enzyme concentration before use, and diluted according to the experimental requirements. The same purified papain (catalogue no P3125; Sigma–Aldrich UK) used in earlier publications (Stepek et al., 2005, 2006, 2007a) was used in the experiments described in this paper. It was diluted in Hanks’ saline to the exact molar concentration of active CPs required for individual in vitro experiments. 2.2. Parasites and hosts We used the rat tapeworm H. diminuta, and the mouse tapeworm H. microstoma, both popular widely used laboratory models of tapeworm infections (Andreassen, 1991; Siles-Lucas and Hemphill, 2002). We refer to H. microstoma rather than Rodentolepis microstoma following Cunningham and Olson (2010). All the animals were housed in groups in standard polypropylene cages containing environmental enrichment, and provided with food and water ad libitum. Five-six week old male Wistar or Lister hooded rats from Charles River were each infected orally by gavage (using a 1 ml syringe with ball tipped needle) with 50 H. diminuta cysticercoids, freshly dissected from infected flour beetles Tribolium confusum. The life cycles of both H. diminuta and H. microstoma have been maintained in Nottingham in a closed colony, using the intermediate host T. confusum beetles, since 1976 but for further details see Behnke (2001a). For maintenance of the life cycles, beetles that had been starved for 4–5 days were given fresh gravid proglottids to consume for 24 h before restoration of their normal diet (48% white flour, 48% wholemeal flour and 4% brewer’s yeast). Cysticercoids were used for infection of animals and for in vitro experiments when at least 28 days old and were suspended in 1 ml of Hanks’ saline. For provision of worms animals were culled on day 14 of infection by a gradually increasing concentration of CO2 , followed by cervical dislocation. We used 14-day old

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worms, which are not yet fully adult (in H. diminuta the pre-patent period is 17 days), but are small enough to fit conveniently into the wells of incubation plates. The small intestine was removed and worms were flushed out with cold Hanks’ saline using a 50 ml syringe. These worms were transferred into a collecting Petri dish containing prewarmed Hanks’ saline at 37 ◦ C before being transferred into individual treatment wells for motility assays or imaging experiments. Male, 6 week old C3H mice from Harlan, U.K. or male BKW mice from B&K U.K. were infected with 12 H. microstoma cysticercoids each, freshly dissected from infected T. confusum, and suspended in 0.2 ml of Hanks’ saline for oral administration. The mice were culled on day 14 of infection by an overdose of CO2 . Because the scoleces of H. microstoma are mainly attached to the bile duct, the liver was removed first from its attachments to the diaphragm and other gut structures in order to expose an intact bile duct. The bile duct was then opened carefully using sharpended dissecting scissors starting proximally from its base at the liver and then moving distally away from the liver towards the duodenum. This incision was then continued in both directions of the duodenum, towards the stomach and towards the ileo-caecal junction. The opened intestine was placed in a glass Petri dish containing pre-warmed Hanks’ saline (37 ◦ C) for 30 min to allow the worms to move away from the tissues. Worms were identified and transferred into another collecting Petri dish containing pre-warmed Hanks’ saline (37 ◦ C) before being transferred to individual treatment wells for motility assay or imaging experiments. To produce cysticercoids of both H. diminuta and H. microstoma, gravid proglottids at the terminal ends of the strobila (∼2 cm) of either H. microstoma or H. diminuta were fed to T. confusum beetles which had been starved for at least 5 days. After 24 h, when the proglottids had been mostly consumed, the beetles were given their regular diet, as detailed above. After 28 days a few representative beetles were dissected to ascertain the presence of fully developed cysticercoids. Fully developed cysticercoids were prepared by artificial excystation for use in juvenile cestode motility assays. This excystation process was based upon an amended protocol by Goodchild and Davis (1972), and Behnke (2001b). Dissected cysticercoids from infected T. confusum beetles were incubated in acidpepsin solution pre-warmed at 37 ◦ C for 15 min in a 37 ◦ C incubator. Acid-pepsin solution was then removed and the cysticercoids were washed three times with Tyrode’s saline. Trypsin-tauroglycocholate solution, pre-warmed at 37 ◦ C, was added and the cysticercoids were incubated at 37 ◦ C for 5 min after which scoleces could be seen leaving their cysts. The authors assert that all procedures contributing to this work comply with the ethical standards of the national guides on the care and use of laboratory animals in the UK (The Animals (Scientific Procedures) Act 1986) and were locally approved by the Animal Welfare and Ethical Review Body of the University of Nottingham. Importantly all work was conducted within a recognised culture of care and compliance to meet the expectations of both the University and the UK Home Office Inspectorate.

2.3. Effect of CPs on pre-adult worm motility In two separate experiments for each species, worms were transferred individually and incubated in 12-well plates (1 worm/well) containing Hanks’ saline (without phenol red), pH 7.2 and 1 mM l-cysteine (Sigma–Aldrich, UK) and one of the following enzyme preparations: 0, 10, 30, 100, 300 ␮M FB, 0, 10, 30, 100, 300, 1000 and 3000 ␮M SB, 0, 10, 30, 100, 300, 1000, 3000 ␮M PLS and 0, 10, 30, 100, 300,1000 ␮M papain. In all cases molarity here refers to that of the functional proteinase active site as determined by active site titration with the specific cysteine proteinase inhibitor E-64 (Apollo Scientific Ltd., UK). The control wells were incubated in parallel and contained: one of the concentrations of enzyme without cysteine (as specified in the results section), no enzyme but with cysteine, enzyme pre-treated with E-64 and cysteine, or Hanks’ saline alone. The worms were incubated at 37 ◦ C for 2 h and their motility was recorded every 15 min using a standard motility scale from 0 to 5 adapted from Stepek et al. (2005) where 0 was assigned to completely motionless worms not responding to manual stimulation, 1 – movement only when prodded, 2 – active only at the ends, i.e. scolex and end of strobila, 3 – slowly spontaneously active, 4 – more active and 5 – highly active. 2.4. Scanning electron microscopy of worms exposed to a variety of plant CPs. In two separate experiments for each species, pre-adult worms were incubated in well plates containing either 1500 ␮M PLS + 1 mM cysteine, 1500 ␮M papain + 1 mM cysteine, 300 ␮M FB + 1 mM cysteine and 2000 ␮M SB + 1 mM cysteine or Hanks’ saline. At 0, 10, 20, and 60 min worms were removed from the wells and placed into bijou bottles containing 2.5% glutaraldehyde in 0.1 M phosphate buffer. After 1 h the glutaraldehyde was removed and replaced with 0.1 M phosphate buffer pH 7.2 and left for another 1 h. The worms were then prepared for scanning electron microscopy (SEM) by further fixation in 1% osmium tetrachloride for 1 h, followed by dehydration in increasing ethanol concentrations from 30 to 100%. They were next subjected to critical point drying with CO2. The dried worms were mounted on disc stubs with quickdrying silver paint and sputter coated with gold before examination in a Jeol JSM 840 Scanning Electron Microscope. 2.5. Effects of CPs on juvenile cestode motility In two separate experiments for each species, fully evaginated scoleces were transferred individually and incubated in 48-well plates (1 worm/well) containing Hanks’ saline (without phenol red), pH 7.2 and 1 mM cysteine and one of the following enzyme preparations: 0, 10, 30, 100, 300 ␮M FB, 0, 10, 30, 100, 300, 1000, 1800, 3000 ␮M PLS, 0, 10, 30, 100, 300, 1000, 1800, 3000 ␮M papain and 0, 10, 30, 100, 300, 1000, 1800 and 3000 ␮M SB. The control wells were incubated in parallel and contained: one of the

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Table 1 Statistical analyses of the effects of CPs on motility of juvenile and adult cestodes. In all cases TIME was fitted as a within subject factor in rmGLM and CONCENTRATION as a between subject factor; all F values are highly significant (P < 0.001). H. diminuta

H. microstoma

CP

Factor

Pre-adult

Juvenile

Pre-adult

Juvenile

FB

TIME CONCENTRATION CONCENTRATION × TIME interaction

F8,80 = 18.5 F4,10 = 159.8 F32,80 = 7.504

F5.5,55.4 = 28.1 F4,10 = 321.7 F22.2,55.4 = 7.7

F8,104 = 132.8 F4,13 = 232.3 F32,104 = 22.7

F7.4,74.1 = 32.3 F4,10 = 106.3 F29.6,74.1 = 8.1

SB

TIME CONCENTRATION CONCENTRATION × TIME interaction

F8,112 = 105.9 F6,14 = 72.0 F48,112 = 11.9

F8,128 = 16.8 F7,16 = 23.7 F56,128 = 3.7

F8,112 = 38.4 F6,14 = 132.1 F48,112 = 9.2

F7.9, 126.1 = 38.7 F7,16 = 180.0 F56,128 = 3.9

PLS

TIME CONCENTRATION CONCENTRATION × TIME interaction

F8,112 = 105.9 F6,14 = 68.5 F48,112 = 9.251

F8,128 = 43.0 F7,16 = 111.5 F56,128 = 8.0

F6.9,96.4 = 38.0 F6,14 = 95.0 F41.3,96.4 = 9.3

F8,128 = 21.7 F7,16 = 14.2 F55.2,126.1 = 7.8

Papain

TIME CONCENTRATION CONCENTRATION × TIME interaction

F8,96 = 24.7 F5,12 = 32.0 F40,96 = 4.396

F8,128 = 24.4 F7,16 = 30.2 F56,128 = 3.6

F5.8,69.7 = 79.2 F5,12 = 99.8 F29.1,69.7 = 8.5

F8,128 = 20.9 F7,16 = 27.0 F56,128 = 4.2

CP = cysteine proteinase; FB = pineapple fruit bromelain; SB = pineapple stem bromelain; PLS = papaya latex supernatant extract.

concentrations of enzyme without cysteine (as specified in the results section), no enzyme but with cysteine, enzyme pre-treated with E-64 and cysteine or Hanks’ saline alone. The worms were incubated at 37 ◦ C for 2 h and their motility was recorded every 15 min as described above.

Mean molity ± S.E.M.

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2.6. Light microscopy of scoleces exposed to a variety of plant CPs In two separate similarly conducted experiments for each species, fully evaginated scoleces (1 worm per well) were transferred into either a well containing

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Time (min) Fig. 1. Motility of pre-adult H. diminuta incubated in four different CPs in vitro; (A) FB; (B) SB; (C) PLS; (D) papain; Hanks’ saline ; Hanks’ saline + 1 mM cysteine 䊉; CP (CP at 300 ␮M in A, 3000 ␮M in B and C, and 1000 ␮m in D) + 1 mM cysteine + E-64 ; CP only (at 300 ␮M in A, 3000 ␮m in B and C, and 1000 ␮M in D) ; 10 ␮M CP + 1 mM cysteine ; 30 ␮M CP + 1 mM cysteine ♦; 100 ␮MCP + 1 mM cysteine ; 300 ␮M CP + 1 mM cysteine  1000 ␮M ; bold trend lines were fitted only for the worms incubated in Hanks’ saline alone and points were CP + 1 mM cysteine X; 3000 ␮M CP + 1 mM cysteine joined for treatments involving both CP and cysteine; error bars represent the standard error of the mean (n = 3) and were plotted for the Hanks’ saline only treatment and for others selectively so as not to obscure data.

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1500 ␮M PLS + cysteine, 300 ␮M FB + cysteine, 1500 ␮M papain + cysteine, 2000 ␮M SB + cysteine or Hanks’ saline.

variable slope and the lower plateau set at 0 and the upper plateau set at the maximum motility for that experiment.

2.7. Statistical analysis 3. Results

Where relevant, data are shown as mean values with standard errors of the mean (S.E.M.). In vitro motility data were analysed using repeated measures general linear models (rmGLM) in SPSS (version 19.0) with TIME after introduction of experimental treatment being fitted as within-subject factor and CONCENTRATION (concentration of PLS, FB, papain and SB) as a between subject factor. The Huynh–Feldt adjustment to the degrees of freedom was used to interpret significance on the side of caution when the data did not meet the requirements of Mauchley’s Test of Sphericity. A P value equal to or less than 0.05 was accepted as indicating a significant difference. All statistical models were checked for approximately normal distribution of residuals. The enzyme concentrations causing 50% reduction in motility (50% inhibitory concentration, IC50 ) values for the motility assays were determined by fitting the data for inhibition of motility, with increasing concentration of CP after 30 min of incubation, in GraphPad Prism 5 using a non-linear regression with

Mean molity ± S.E.M.

6

3.1. Efficacy of CPs against pre-adult cestodes The motilities of adult H. diminuta and H. microstoma declined significantly with time of incubation when incubated in all the CPs tested (Table 1, Figs. 1 and 2). Motility was rapidly reduced in the presence of higher concentrations of active CPs but was not dependent on the presence of exogenous cysteine in the incubation medium (Figs. 1 and 2) and showed significant CP concentration dependent inhibition of motility (Table 1 and Fig. 3). However, motility was relatively unaffected when worms were incubated in the presence of low concentrations of active CPs or in the control solutions of Hanks’ saline with or without cysteine and also in the presence of CP pre-treated with E-64 (Fig. 1 and Fig. 2). Although all CPs tested reduced motility, FB was the most potent on both species and SB was the least effective. (Fig. 3 and Table 2)

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Time ( min) Fig. 2. Motility of pre-adult H. microstoma incubated in four different CPs in vitro; (A) FB; (B) SB; (C) PLS; (D) papain; Hanks’ saline ; Hanks’ saline + 1 mM cysteine 䊉; CP (CP at 300 ␮M in A, 3000 ␮M in B and C, and 1000 ␮M in D) + 1 mM cysteine + E-64 ; CP only (at 300 ␮M in A, 3000 ␮M in B and C, and 1000 ␮M in D) ; 10 ␮M CP + 1 mM cysteine ; 30 ␮M CP + 1 mM cysteine ♦; 100 ␮M CP + 1 mM cysteine ; 300 ␮M CP + 1 mM cysteine  1000 ␮M ; bold trend lines were fitted only for the worms incubated in Hanks’ saline alone and points were CP + 1 mM cysteine X; 3000 ␮M CP + 1 mM cysteine joined for treatments involving both CP and cysteine; error bars represent the standard error of the mean (n = 3) and were plotted for the Hanks’ saline only treatment and for others selectively so as not to obscure data.

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Fig. 3. Concentration–inhibition curves of the effects of four CPs on motility of pre-adult H. diminuta (A) and pre-adult H. microstoma (B), artificially excysted H. diminuta (C) and H. microstoma (D) after 30 min incubation. Points are the mean ± S.E.M. Lines were fitted using a non-linear regression with variable slope in Graphpad Prism 5. FB ; PLS ; Papain ; SB ♦.

3.2. SEM of cestodes incubated in CPs Progressive changes to the cestode tegument were observed in worms incubated in PLS, papain and SB, reflected in blunting of the proglottid edges and a generally eroded appearance that became apparent even at 10 min post incubation (Fig. 4A, D and J). After 20 min there was deeper erosion (Fig. 4B, E and K). After 60 min post incubation the erosion became so extensive as to expose the internal proglottid contents (Fig. 4C, F and L). Worms incubated in FB, however, showed different changes to the tegument, albeit the presence of similar very superficial erosion was still apparent. At 10 min post incubation there was formation of transverse wrinkling (Fig. 4G) and at 20 min post incubation only slight erosion and blunting of the proglottid edges (Fig. 4H). At 60 min post incubation apart from extensive transverse wrinkling and blunted proglottid edges, there were also pockets which appeared

to be fluid filled blebs on the worm’s lateral edges (Fig. 4I). The worms incubated in Hanks’ saline, however, did not show any significant change on the tegument surface even after 60 min of incubation (Fig. 4N). When imaging was focused on the worm’s scolex, similar progressive erosion was apparent but this was comparatively less intense (Fig. 5A–C). Similarly, in H. microstoma, progressive changes were observed on the tegumental surface beginning with blunting of proglottids, noted at 10 min post incubation (Fig. 6A) and developing into a generalised superficial eroded appearance by 20 min (Fig. 6B). At 60 min there was marked generalised erosion and at some points tegumental discontinuity was observed with the release of internal contents (Fig. 6C). The worms incubated in Hanks’ saline however, maintained the same appearance even after 60 min with sharp proglottid edges and a smooth tegumental surface (see Fig. 6D and E).

Table 2 IC50 values for juvenile and adult tapeworms incubated in different plant-derived cysteine proteinase solutions for 30 min. Values were obtained from concentration inhibition curves shown in Fig. 3. IC50 value of CP (␮M) Cestode species

Stage

FB

SB

PLS

Papain

H. diminuta H. diminuta H. microstoma H. microstoma

Pre-adult Juvenile Pre-adult Juvenile

199 63 260 74

1374 2855 1332 2772

818 943 776 984

892 2454 857 2316

CP = cysteine proteinase; FB = pineapple fruit bromelain; SB = pineapple stem bromelain; PLS = papaya latex supernatant extract.

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Fig. 4. Scanning electron micrographs of pre-adult H. diminuta incubated in various CPs in vitro. A–C in PLS 1500 ␮M + Hanks’ saline + 1 mM cysteine; D–F in papain 1500 ␮M + Hanks’ saline + 1 mM cysteine; G–I, in FB 300 ␮M + Hanks’ saline + 1 mM cysteine; J–L, in SB 2000 ␮M + Hanks’ saline + 1 mM cysteine, M and N; in Hanks’ saline. Note the obvious erosion of the tegument in PLS, papain, SB after 60 min (C, F and L) compared with the lateral swelling in FB (I) Scale bars = 100 ␮m.

3.3. Effects of CPs on juvenile cestodes As with the pre-adult worms, the motility of artificially excysted H. diminuta and H. microstoma scoleces also declined significantly with time of incubation in CPs (Table 1) and showed a rapid reduction in motility in the presence of higher concentrations of active CPs but was not dependent on the presence of exogenous cysteine in the incubation medium (Figs. 7 and 8). Increasing concentrations of active CPs reduced motility in both species (Table 1 and Fig. 3). Motility was unaffected when artificially excysted scoleces were incubated in the presence of low concentrations of active CPs or in the control solutions of Hanks’ saline with or without cysteine and in the presence of CP pre-treated with E-64 (see Figs. 7 and 8). Although all CPs tested reduced motility, FB was the most

potent on both species and SB was the least effective. (Fig. 3 and Table 2) 3.4. Light microscopy of juvenile cestodes incubated in CPs After one hour of incubation in active CPs, there were notable changes in morphology of H. diminuta freshly excysted metacestodes (results not illustrated) with apparent ballooning of the distal end of worms incubated in PLS (1500 ␮M + 1 mM cysteine), papain (2000 ␮M+ 1 mM cysteine) and SB (2000 ␮M + 1 mM cysteine). The worms incubated in FB (300 ␮M + 1 mM cysteine) however appeared to shrink and contract. After two hours the worms incubated in PLS appeared to be partially digested while

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Fig. 5. Pre-adult H. diminuta incubated in PLS in vitro focusing on the scolex only. A–C; in PLS 1500 ␮M + Hanks’ saline + 1 mM cysteine; D and E in Hanks’ saline. In the presence of PLS, note the progressive erosion of the scolex tegument and the collapse of the globular shape by 60 min while the scolex was perfectly preserved in Hanks’ saline even after 60 min. Scale bars = 10 ␮m.

those incubated in FB remained unchanged in the contracted state that had been observed after one hour and those incubated in papain appeared to develop shrinkage of the neck region. No significant changes in morphology were observed in the worms incubated in Hanks’ saline. Very similar changes were observed on H. microstoma. 4. Discussion CPs have already been shown to have potent anthelmintic effects on a range of adult intestinal nematode parasites (Stepek et al., 2005, 2006, 2007b) and to have some effect on juvenile plant parasitic nematodes (Stepek et al., 2007a). Now our data extend these studies showing clearly that the efficacy of CPs is not limited to nematodes alone. CPs, whether naturally occurring mixtures such as papaya latex and relatively pure preparations such as FB, SB and papain caused significant reductions in motility leading to death of both species of rodent cestodes

in vitro, irrespective of whether the worms were juveniles or pre-adults. The reduction in motility on exposure to active CPs was also concentration dependant as has been reported previously for nematodes (Stepek et al., 2005). However, there were some striking differences between our observations on cestodes and earlier published reports on nematodes. Firstly, the reductions in motility mediated by CPs against cestodes were independent of the presence of the exogenous CP activator cysteine, while inclusion of cysteine has been crucial for all the nematodes studied in vitro to-date (Stepek et al., 2005). Cysteine acts as a reducing agent and the active site on CPs requires activation through reduction before the enzymes can digest their target molecules (Storer and Ménard, 1994). The cestode tegument is known to be highly active metabolically and enzymatically (Schardein and Waitz, 1965; Lumsden, 1966; Öhman-James, 1973) and its surface is believed to be polyionic (Williams and Hoole, 1995), facilitating the adhesion of a variety of host proteins. Some of the charged molecules may well have acted as reducing agents and

Fig. 6. Adult H. microstoma incubated in PLS 1000 ␮M + Hanks’ saline + 1 mM cysteine (A–C) showing clear damage to the tegument after 60 min and undamaged worms in Hanks’ saline (D and E). Scale bars = 100 ␮m.

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A.

Mean molity ± S.E.M.

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Time (min) Fig. 7. Motility of artificially excysted scoleces H. diminuta incubated in various CPs in vitro (A) FB, (B) SB, (C) PLS, (D) papain; Hanks’ saline ; Hanks’ saline + 1 mM cysteine 䊉; CP (CP at 300 ␮M in A, 3000 ␮M in B, C, and D) + 1 mM cysteine + E-64 ; CP only (at 300 ␮M in A, 3000 ␮M in B, C and D) ; 10 ␮M CP + Hanks’ saline + 1 mM cysteine ; 30 ␮M CP + Hanks’ saline + 1 mM cysteine ♦; 100 ␮M CP + Hanks’ saline + 1 mM cysteine ; 300 ␮M CP + Hanks’ saline + 1 mM cysteine  1000 ␮M CP + Hanks’ saline + 1 mM cysteine X; 1800 ␮M CP + Hanks’ saline + 1 mM cysteine ∗; 3000 ␮M CP + Hanks’ saline + 1 mM ; bold trend lines were fitted only for the worms incubated in Hanks’ saline alone and points were joined for treatments involving both CP cysteine and cysteine; error bars represent the standard error of the mean (n = 3) and were plotted for the Hanks’ saline only treatment and for others selectively so as not to obscure data.

activated the CPs in our study. Moreover, as with all helminths, cestodes naturally shed material from their surface layers and excrete/secrete various molecules from glands, such as for example the apical rostellar glands of Echinoccocus granulosus (Thompson et al., 1979), among which also there may be suitable reducing agents capable of activating CPs by donating electrons. E. granulosus secretes a globular protein that is rich in cysteine Thompson et al. (1979) and H. diminuta is known to secrete hydrogen ions making the medium surrounding the worms acidic (Podesta and Mettrick, 1974), a process that is likely in turn to leave negatively charged ions in association with the parasite’s tegument. These possibilities could be examined further. Secondly, the concentrations of CPs that caused significant loss of motility were considerably higher than those reported for nematodes, although in Stepek et al. (2005) a longer incubation time was employed, IC50 values being calculated after 90 min of incubation. However, more recently Luoga et al. (2014) calculated IC50 values for the intestinal nematode Heligmosomoides bakeri after 30 min of incubation and for PLS this was just 67.8 ␮M compared with values of at least 776 ␮M (Table 2) for cestodes in the current study. For FB Luoga et al. (2014) recorded values of 33.0–96.1 ␮M which is in the same

range as those of juvenile cestodes in the current study but lower than those for pre-adult cestodes. Values for SB were 150–1142 ␮M, but still lower than those for both stages of both cestodes here. Clearly cestode teguments are more resistant to attack by CPs than nematode cuticles and in part this may be because of a more limited availability of susceptible digestion sites. However, another possible explanation may lie in the cestodes’ extraordinary ability to recycle tegumental membranes and to repair damage (Oaks and Lumsden, 1971; Befus and Threadgold, 1975; Hoole and Arme, 1985). This capacity to rapidly repair and thereby keep pace with host response-induced damage to their surface has been offered as an explanation for the long-term survival of cestodes in their definitive hosts (McCaigue et al., 1986). It may also be significant that, with the exception of FB, we found that the excysted juvenile worms were more resistant to attack by SB, PLS and papain. This may be related to the generally greater resistance of cestode scoleces to immune attack (Andreassen and Hoole, 1989) and treatment with anthelmintics (Dixon and Arai, 1991), both of which may be attributed to differences in the composition and properties of the tegument (and its glycocalyx layer) in the region of the scolex and neck compared with the strobila (Taylor et al., 1997).

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Time (min) Fig. 8. Motility of artificially excysted H. microstoma scoleces incubated in various CPs in vitro (A) FB; (B) SB; (C) PLS; (D) papain; Hanks’ saline ; Hanks’ + 1 mM Hanks’ cysteine 䊉; CP (CP at 300 ␮M in A, 3000 ␮M in B, C, and D) + 1 mM cysteine + E-64 ; CP only (at 300 ␮M in A, 3000 ␮M in B, C and D) ; 10 ␮M CP + Hanks’ saline + 1 mM cysteine ; 30 ␮M CP + Hanks’ saline + 1 mM cysteine ♦; 100 ␮M CP + Hanks’ saline + 1 mM cysteine ; 300 ␮ M CP + Hanks’ saline + 1 mM cysteine  1000 ␮M CP + Hanks’ saline + 1 mM cysteine X; 1800 ␮M CP + Hanks’ saline + 1 mM cysteine ∗; 3000 ␮M CP + Hanks’ ; bold trend lines were fitted only for the worms incubated in Hanks’ saline alone and points were joined for treatments saline + 1 mM cysteine involving both CP and cysteine; error bars represent the standard error of the mean (n = 3) and were plotted for the Hanks’ saline only treatment and for others selectively so as not to obscure data.

Various CPs have been screened already for their efficacy against H. bakeri in vitro ranging from naturally occurring mixtures such as papaya latex and milkweed latex to relatively pure preparations such as ficin, actinidain, papain, chymopapain, FB and SB (Stepek et al., 2005). The efficacy of various CPs against H. bakeri varied considerably; with papain and chymopapain ranking as the most potent while actinidain was not at all efficacious (Stepek et al., 2005). However, here it was evident in both rodent cestodes and with respect to both the pre-adult and juvenile stages tested that FB was the most potent (see Table 2) and this was followed by PLS, then papain with SB being the least effective in reducing motility. It was apparent also that FB was considerably more potent in reducing motility than the other enzymes tested although this was not reflected in the observable damage to the tegument. In fact, the changes in the tegument in FB appeared different from those due to the other CPs as observed under SEM, which may relate to the underlying mechanism in terms of the target protein(s) in the tegument. In worms incubated in FB there appeared to be no marked tegumental erosions (Fig. 4G–I) as was observed in worms incubated in other CPs such as PLS (Figs 4A–C), papain (Fig. 4D–F) and SB (Fig. 4J–L). Although the potency of different types of CPs against the same cestodes varied, it did not differ substantially between the two different rodent cestodes tested, whether pre-adults or juvenile stages, and was comparable in both

worms albeit with slightly greater activity against H. diminuta. In summary, the results from the experiments described in this paper indicate that CPs are indeed efficacious against cestodes in vitro by significantly reducing their motility and causing death of the worms. In contrast to the effects on nematodes, CPs are able to exert their proteolytic activity against cestodes in vitro without the presence of exogenous cysteine. Despite the limited nature and the uncertainty surrounding published reports on CP efficacy against juvenile stages of nematodes, (Stepek et al., 2006, 2007c), CPs were indeed efficacious against juvenile cestodes. Natural mixtures of CPs appeared to be more effective than purified CPs and as in nematode cuticles, appeared to digest the tegument giving rise to an eroded appearance that was evident from SEM images but which was quite different from the known modes of action of conventional cestocidals. Acknowledgements FM was supported by a postgraduate studentship from the government of Malaysia, and WL from the Government of Tanzania. We thank Tim Smith for technical support with electron microscopy. We dedicate this paper to Dr. Wenceslaus Luoga who sadly was killed in a road accident in July 2013 shortly after returning to Tanzania on

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the successful completion of his post-graduate studies at the University of Nottingham References Andreassen, J., 1991. Immunity to adult cestodes: basic knowledge and vaccination problems. A review. Parassitologia 33, 45–53. Andreassen, J., Hoole, D., 1989. Repair of Hymenolepis diminuta after complement-mediated damage. Parasitology 99, 437–443. Barrett, A.J., Kembhavi, A.A., Hanada, K., 1981. E-64 [L-transand related epoxysuccinyl-leucyl-amido(4-guanidino)butane] epoxides as inhibitors of cysteine proteinases. Acta biologica et medica Germanica 40, 1513–1517. Befus, A.D., Threadgold, L.T., 1975. Possible immunological damage to the tegument of Hymenolepis diminuta in mice and rats. Parasitology 71, 525–534. Behnke, J.M., 2001a. Hymenolepis species (Cestoda). In: Halton, D.W., Behnke, J.M., Marshall, I. (Eds.), British Society for Parasitology. Practical Exercises in Parasitology. , pp. 115–121. Behnke, J.M., 2001b. Activation of the cysticercoids of Hymenolepis species in vitro. In: Halton, D.W., Behnke, J.M., Marshall, I. (Eds.), British Society for Parasitology. Practical Exercises in Parasitology. , pp. 183–190. Behnke, J.M., Buttle, D.J., Stepek, G., Lowe, A., Duce, I.R., 2008. Developing novel anthelmintics from plant cysteine proteinases. Parasites & Vectors 1, 29. Buttle, D.J., Dando, P.M., Coe, P.F., Sharp, S.L., Shepherd, S.T., Barrett, A.J., 1990. The preparation of fully active chymopapain free of contaminating proteinases. Biol. Chem. Hoppe-Seyler 371, 1083–1088. Buttle, D.J., Behnke, J.M., Bartley, Y., Elsheikha, H.M., Bartley, D.J., Garnett, M.C., Donnan, A.A., Jackson, F., Lowe, A., Duce, I.R., 2011. Oral dosing with papaya latex is an effective anthelmintic treatment for sheep infected with Haemonchus contortus. Parasites & Vectors 4, 36. Cox, F.E., 2004. History of human parasitic diseases. Infect. Dis. Clin. North Am. 18, 171–188. Cunningham, L.J., Olson, D.P., 2010. Description of Hymenolepis microstoma (Nottingham, strain): a classical tapeworm model for research in the genomic era. Parasites & Vectors 3, 123. de Amorin, A., Borba, H.R., Carauta, J.P., Lopes, D., Kaplan, M.A., 1999. Anthelmintic activity of the latex of Ficus species. J. Ethnopharm. 64, 255–258. Dixon, B.R., Arai, H.P., 1991. Anthelmintic-induced destrobilation and its influence on calculated drug efficacy in Hymenolepis diminuta infections in rats. J. Parasitol. 77, 769–774. Ghosh, N.K., Babu, S.P., Sukul, N.C., Ito, A., 1996. Cestocidal activity of Acacia auriculiformis. J. Helminthol. 70, 171–172. Goodchild, C.G., Davis Jr., B.O., 1972. Hymenolepis microstoma cysticercoid activation and excystation in vitro (Cestoda). J. Parasitol. 58, 735–741. He, S., Tiuria, R., Retnani, B.E., 1992. Uji biologis aktivitas anthelmintik sari buah nanas muda, daun miana dan ranting puring terhadap cacing Aspiculuris tetraptera (Nematoda) dan Hymenolepis nana pada mencit putih (Mus musculus albinus). Hemera Zoa. 75, 94–110. Hoole, D., Arme, C., 1985. The in vitro culture and tegumental dynamics of the plerocercoid of Ligula intestinalis (Cestoda: Pseudophyllidea). Int. J. Parasitol. 15, 609–615. Kundu, S., Lyndem, L.M., 2012. In vitro screening for cestocidal activity of three species of Cassia plants against the tapeworm Raillietina tetragona. J. Helminthol. 87, 154–159. Kundu, S., Roy, S., Lyndem, L.M., 2012. Cassia alata L: potential role as anthelmintic agent against Hymenolepis diminuta. Parasitol. Res. 3, 1187–1192. Luoga, W., Mansur, F., Buttle, D.J., Duce, I.R., Garnett, M.C., Lowe, A., Behnke, J.M., 2014. The relative anthelmintic efficacy of plant-derived cysteine proteinases on intestinal nematodes. J. Helminthol., http://dx. doi.org/10.1017/S0022149X13000692 (Epub ahead of print). Lumsden, R.D., 1966. Cytological studies on the absorptive surfaces of cestodes II the synthesis and intracellular transport of protein in the strobilar integument of Hymenolepis diminuta. Z. Parasitenkd. 28, 1–13.

McCaigue, M.D., Halton, D.W., Hopkins, C.A., 1986. Hymenolepis diminuta: ultrastructural abnormalities in worms from C57 mice. Exp. Parasitol. 62, 51–60. Oaks, J.A., Lumsden, R.D., 1971. Cytological studies on the absorptive surfaces of cestodes. V. Incorporation of carbohydrate-containing macromolecules into tegument membranes. J. Parasitol. 57, 1256–1268. Öhman-James, C., 1973. Cytology and cytochemistry of the scolex gland cells in Diphyllobothrium ditremum (Creplin, 1825). Z. Parasitenkd. 42, 77–86. Podesta, R.B., Mettrick, D.F., 1974. The effect of bicarbonate and acidification on water and electrolyte absorption by the intestine of normal and infected (Hymenolepis diminuta: Cestoda) rats. Am. J. Dig. Dis. 19, 725–735. Prakash, V., Mehrotra, B.N., 1987. Anthelmintic plants in traditional remedies in India. Indian J. Hist. Sci. 22, 332–340. Rowan, A.D., Buttle, D.J., Barrett, A.J., 1990. The cysteine proteinases of the pineapple plant. Biochem. J. 266, 869–875. Schardein, J.L., Waitz, J.A., 1965. Histochemical studies of esterases in the cuticle and nerve cords of four cyclophyllidean cestodes. J. Parasitol. 51, 356–363. Siles-Lucas, M., Hemphill, A., 2002. Cestode parasites: application of in vivo and in vitro models for studies on the host-parasite relationship. Adv. Parasitol. 51, 133–230. Stepek, G., Buttle, D.J., Duce, I.R., Lowe, A., Behnke, J.M., 2005. Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro. Parasitology 130, 203–211. Stepek, G., Lowe, A.E., Buttle, D.J., Duce, I.R., Behnke, J.M., 2006. In vitro and in vivo anthelmintic efficacy of plant cysteine proteinases against the rodent gastrointestinal nematode. Trichuris muris. Parasitol. 132, 681–689. Stepek, G., Curtis, R.H., Kerry, B.R., Shewry, P.R., Clark, S.J., Lowe, A.E., Duce, I.R., Buttle, D.J., Behnke, J.M., 2007a. Nematicidal effects of cysteine proteinases against sedentary plant parasitic nematodes. Parasitology 134, 1831–1838. Stepek, G., Lowe, A.E., Buttle, D.J., Duce, I.R., Behnke, J.M., 2007b. Anthelmintic action of plant cysteine proteinases against the rodent stomach nematode, Protospirura muricola, in vitro and in vivo. Parasitology 134, 103–112. Stepek, G., Lowe, A.E., Buttle, D.J., Duce, I.R., Behnke, J.M., 2007c. The anthelmintic efficacy of plant-derived cysteine proteinases against the rodent gastrointestinal nematode, Heligmosomoides polygyrus, in vivo. Parasitology 134, 1409–1419. Storer, A.C., Ménard, R., 1994. Catalytic mechanism in papain family of cysteine peptidases. Methods Enzymol. 244, 486–500. Tandon, V., Das, B., Saha, N., 2003. Anthelmintic efficacy of Flemingia vestita (Fabaceae): effect of genistein on glycogen metabolism in the cestode, Raillietina echinobothrida. Parasitol. Int. 52, 179–183. Tangpu, V., Temjenmongla, Yadav, A.K., 2006. Anticestodal property of Strobilanthes discolor: an experimental study in Hymenolepis diminuta–rat model. J. Ethnopharm. 105, 459–463. Taylor, K., Kusel, J.R., Hoole, D., 1997. Membrane fluidity on the surface of Hymenolepis diminuta (Cestoda): immunological implications. Parasitol 115, 667–671. Thompson, R.C.A., Dunsmore, J.D., Hayton, A.R., 1979. Echinococcus granulosus: secretory activity of the rostellum of the adult cestode in situ in the dog. Exp. Parasitol. 48, 144–163. Waller, P.J., Bernes, G., Thamsborg, S.M., Sukura, A., Richter, S.H., Ingebrigtsen, K., Hoglund, J., 2001. Plants as de-worming agents of livestock in the Nordic countries: historical perspective, popular beliefs and prospects for the future. Acta Vet. Scand. 42, 31–44. Williams, M.A., Hoole, D., 1995. Immunolabelling of fish host molecules on the tegumental surface of Ligula intestinalis (Cestoda: Pseudophyllidea). Int. J. Parasitol. 25, 249–256. Yadav, A.K., Tangpu, V., 2008. Anticestodal activity of Adhatoda vasica extract against Hymenolepis diminuta infections in rats. J. Ethnopharm. 119, 322–324. Zucker, S., Buttle, D.J., Nicklin, M.J.H., Barrett, A.J., 1985. The proteolytic activities of chymopapain, papain, and papaya proteinase III. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 828, 196–204.