Zeitschrift ffir

Parasitenkunde

Z. Parasitenkd. 63,261-269 (]980)

Parasitology Research

9 by Springer-Verlag 1980

A New Primary Screening Test for Anthelmintics Utilizing the Parasitic Stages of Nippostrongylus brasiliensis, in vitro D.C. Jenkins, R. Armitage, and T.S. Carrington Department of Parasitology, The Wellcome Research Laboratories, Beckenham, Kent, England

Abstract. A new in vitro test suitable for the large scale screening of chemical compounds for anthelmintic activity is described. The test which utilizes the fourth larval and adult stages of Nippostrongylus brasiliensis in a medium capable of supporting the growth and development of the parasite, detects selectively those compounds which possess either broad spectrum anthelmintic or specific anti-trichostrongyle activity. The screen is easy to operate requiring only minute quantities of experimental compound. It renders fully reproducible results which furthermore can be interpreted objectively. This is the first reported in vitro test directed against the parasitic stages of a nematode that is capable of detecting reliably the activity of a wide range of anthelmintics including thiophanate and all the benzimidazoles. Key words: In vitro culture - Anthelmintics - Primary screening test -

Nippostrongylus brasiliensis.

Introduction The paucity of our knowledge of the biochemistry of parasitic nematodes still largely mitigates against the exclusive use of rational screening tests for the discovery of novel broad spectrum veterinary anthelmintics. The empirical screening of as wide a range as possible of chemical compounds against these parasites continues therefore to be one of our best means of achieving this goal. To attempt such screening in the target host, e.g. sheep or cattle, is prohibitively expensive in terms of labour and materials and even tests against related parasites in small rodents demand too much experimental compound which is often available only in very small quantities. The need for rapid repeatable and relevant in vitro screening tests for the discovery of new leads is therefore obvious. In vitro screens using free-living nematodes (Tiner, 1958; Vanfleteren and Roets, 1972; Platzer et al., 1977) have been in use for many years as have

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tests a g a i n s t the free-living stages o f p a r a s i t i c n e m a t o d e s . T i n e r (1958) used the eggs a n d free-living stages o f t r i c h o s t r o n g y l i d w o r m s f r o m r a b b i t s , a n d Coles a n d M c N e i l l i e (1977) e m p l o y e d a test using the eggs o f Nematodirus spathiger. W e have used similar tests b u t in general find t h e m to be t o o sensitive giving rise to an u n a c c e p t a b l y large n u m b e r o f irrelevant " l e a d s " F e w in vitro screening tests e m p l o y i n g the p a r a s i t i c stages o f t r i c h o s t r o n g y l i d n e m a t o d e s are r e p o r t e d in the literature. L e l a n d et al. (1975) used the n e m a t o d e , Cooperia punctata in a c o m p l e x culture m e d i u m with s o m e success. H o w e v e r , the test was c u m b e r s o m e to o p e r a t e r e q u i r i n g l o n g p e r i o d s of i n c u b a t i o n . F u r ther, their p u b l i s h e d d a t a d o n o t indicate w h e t h e r their test was sufficiently selective for the specific d e t e c t i o n o f a n t h e l m i n t i c c o m p o u n d s . Coles and M c N e i l l i e (1977) h a v e d e s c r i b e d a test using the a d u l t stage o f the r a t t r i c h o s t r o n gyle, Nippostrongylus brasiliensis, in vitro. H o w e v e r , this test was carried o u t in a m e d i u m which merely m a i n t a i n e d the w o r m s in a live state for a s h o r t period. The test was useful for the d e t e c t i o n o f activity in s o m e c o m p o u n d s such as the i m i d a z o t h i a z o l e s a n d the p y r i m i d i n e s b u t failed to d e t e c t s o m e o t h e r anthelmintics, n o t a b l y the b e n z i m i d a z o l e s . The present p a p e r describes a new m o r e r e l e v a n t in vitro screening test a g a i n s t the p a r a s i t i c f o u r t h larval and a d u l t stages o f N. brasiliensis.

Material and Methods

Culture Medium The medium used is described in detail in Table 1. This was a modified version of the medium used previously by Keeling et al. (1980) for the culture of the nematode, Nippostrongylusbrasiliensis from the infective stage to the adult. Modifications were the use of calf serum rather than rat

Table 1. Composition of the culture medium Tryptic digest of casein (Difco) Yeast Extract (Difco or B.B.L.) D-glucose Di-potassium hydrogen orthophosphate Potassium dihydrogen orthophosphate Distilled water . . . . . . add to Adjust pH to 7.2 with 10 N KOH Autoclave at 20 1b for 15 min

2.0 g 1.0 g 0.5 g 0.08 g 0.08 g 100.0 ml

Sodium benzylpenicillin Streptomycin sulphate 5-fluorocytosine Calf serum (heat inactivated) Bovine erythrocyte lysate (Blood cells lysed in an equal volume of sterile water)

60 mg 100 mg 1 mg 18 ml 2.4 ml

Final pH=7.2 Osmolality= 268 milliosmoles

Basal medium Complete medium

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serum, bovine erythrocyte lysate instead of rat erythrocyte lysate, the inclusion of the c o m p o u n d 5-fluorocytosine to control yeasts, and the omission of a vitamin supplement.

Preparation of Worms for Culture Fourth stage parasitic larvae of N. brasiliensis were used at the c o m m e n c e m e n t of each experiment. These were obtained from the intestines of 8-week-old, category 2, Sprague-Dawley rats infected three days previously each with 5,000 infective larvae. The worms were collected at 3 7 ~ in glucose Tyrode solution containing the antibiotics, sodium benzylpenicillin (0.12%), streptomycin sulphate (0.2%), and the anti-yeast agent, 5-fluorocytosine (0.001%). After collection, the worms were incubated at 37~ for further 2 h in this solution, with two changes in order to clean the worms prior to culture. Finally the worms were resuspended at a concentration of 250 worms per mI in complete medium ready for the test.

Formulation of Test Compounds All c o m p o u n d s were treated by a standard method devised for the formulation of compounds of extreme insolubility. Five milligrammes of the c o m p o u n d were placed in a sterile screw capped 30 ml bottle to which 0.1 ml of ethanol was added to dissolve or, if insoluble, wet, and sterilize the compound. A 10% solution of complete culture medium in water was then added to give a final volume of 10 ml. The bottle containing the resultant solution/suspension was then placed in a sonic water bath (Sonicor Ltd.) and sonicated for 10 min at 23 ~ C. Further dilutions were then made with sterile water, the solution/suspension being thoroughly dispersed with the aid of a " W h i r l i m i x e r " (Fisons Ltd.) during each dilution.

Operation of the Test for Evaluating Known Anthelminties During the early part of the work, ai1 tests were carried out in glass Leighton tubes. To each tube, 1.6 ml of complete medium was added followed by 0.2 ml of appropriately diluted drug solution/suspension. A further 0.2 ml sample of medium containing 50 worms was then added to each tube giving a final volume of 2.0 ml. K n o w n anthelmintics were tested at concentrations ranging from 1,000.0 to 0.01 ~tg per ml. In each experiment each drug concentration was set up in duplicate with an untreated control group of six tubes. For incubation, the tubes were closed with a screw cap and placed on ' M a t b u r n ' rollers operating at 0.2 rpm in a dark room at 37 ~ C. The duration of each test was seven days, the worms being examined daily under an inverted microscope (Zeiss, "Invertoscope D"). The medium was not changed during the experiments. After the standard anthelmintics had been tested on several occasions, the test was modified, the Leighton tubes being replaced by multichambered plastic dishes ( " R e p l i " - Sterilin Ltd.). These proved more convenient in use and had no adverse effects on the performance of the test. No change was made in the volume of m e d i u m or the n u m b e r of worms used per test. However, for incubation the Repli dishes were not placed on any rolling or shaker device, a n u m b e r of trial runs showing that the development of the worms in dishes left stationary on shelves in the incubator was in no way impeded.

Interpretation of the Tests Activity of the compounds was assessed by comparing the rate of development and the motility of the test worms with that of the controls. The major parameter for assessing activity was whether the moult from fourth larval (L4) to the adult stage occurred. For each compound tested the percentage moult was determined, this being calculated from data on the m e a n n u m b e r of cast cuticles found in the test chambers relative to the controls.

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Results

Development of the Worms under the Conditions of the Screening Test Prior to testing known anthelmintic compounds in this screen, the course of development of the worms under conditions identical with those in the untreatedcontrol tubes was examined. In each experiment, eight tubes each containing 50 worms were used. The results of a typical experiment to determine the mean length of the worms on days 0 and 7, and the percentage of worms moulting from the fourth larval stage to adults during that period are given in Table 2.

The Activity of Known Anthelmintics The minimum inhibitory concentrations of each of the anthelmintics tested and their effects on moulting are given in Table 3. Avermectin Bla, laevamisole, morantel tartrate, thiophanate, and all the benzimidazoles were highly active in the test whilst amoscanate, nitroscanate, and phenothiazine showed moderate but significant activity. Bephenium was only weakly active and piperazine, diethylcarbamazine, and suramin were inactive. At effective concentrations (Fig. 1 a-c) laevamisole, morantel tartrate, and the isothiocyanates exerted their effects rapidly, the worms becoming tightly coiled and immobilized before day 1. None of the worms incubated with these compounds moulted from the L4 to the adult stage and all the worms were dead by day 3. Their structure was grossly disorganised, no discreet internal organs being discernible. The effects of the benzimidazole compounds and thiophanate on the worms (Fig. 1 d) were more gradual. By day 3, the worms were very sluggish of move-

T a b l e 2. Nippostrongylus brasiliensis : D e v e l o p m e n t o f the w o r m s u n d e r the c o n d i t i o n s of the test

Day of test

0 1

L4 F o u r t h stage l a r v a e a E a c h g r o u p consisted o f 20 w o r m s b E a c h g r o u p consisted of eight tubes each c o n t a i n i n g 50 w o r m s

2 3 4 5 6 7

M e a n w o r m length m m . +_ S . D . " r a n g e given in b r a c k e t s

1.70_+0.29 (1.41 - 1.99) -

-2.50_+0.26 (2.23 - 2.76)

2.04_+0.51 (1.52 - 2.54) --

---2.57+0.48 (2.09 - 3.05)

% moulted f r o m L4 to a d u l t -+ S.D. b

0 0

45.2+26.25 66.2_+21.31 75.0 _+ 18.73 78.7 -+ 19.24 84.5+13.55 86.7-+12.32

An in vitro Screening Test for Anthelmintics Table 3. In vitro Activity of known anthelmintics against the parasitic stages of Nippostrongylus brasiliensis

265

Compounds

Minimum inhibitory concentration p_g/ml

a

L 4 Fourth stage larvae

gM

Thiabendazole 0.05 0.25 Cambendazole 0.05 0.17 Parbendazole 0.05 0.20 Mebendazole 0.05 0.17 Fenbendazole 0.I 0.33 Oxfendazole 0.1 0.32 Oxibendazole 0.0t 0.04 Flubendazole 0.1 0.33 Albendazole 0.05 0.19 Thiophanate 0.5 1.30 Avermectin Bla < 0.01 < 0.0l Laevamisole 0.5 2.08 hydrochloride Morantei tartrate 0.5 1.55 Phenothiazine 5.0 25.10 Bephenium 100.0 225.7 hydroxy naphthoate Piperazine > 1,000.0 hexahydrate Diethyl> 1~000.0 carbamazine citrate Suramin > 1,000.0 Amoscanate 5.0 19 45 Nitroscanate 10.0 36.72 Untreated controls -

% moult from L4 a to adult stage 0 0 0 0 0 0 0 8 0 0 12 0 0 6 77

> 85 > 85

> 85 0 0 >85

ment compared with the controls. The development of the worms was also inhibited, none attaining the adult stage. By day 7 most of the worms had died, the few survivors being almost immotile and obviously arrested at the L, stage of development. Like the benzimidazoles, the effects of the macrolide, avermectin Bla were gradual. Very few worms succeeded in moulting to the adult stage, both these and the inhibited L 4 worms becoming very sluggish by day4. Only a few worms were actually dead by day 7 but the survivors were virtually immotile, very pale in colour, and of grossly abnormal appearance (Fig. 1 e). Phenothiazine (Fig. 1 f) showed marked inhibitory effects on the development and motility of the worms. These effects were clearly evident by day 2 but did not seem to become more pronounced with time, the worms having a similar appearance, at the light microscope level, on day 7. The weak activity of bephenium was manifest only by some inhibition of motility and the death of some worms by day 7. The gross appearance of the surviving worms was not significantly different from that of the controls. These controls are shown on Fig. 2a-c.

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Fig. 1 a-f. Effects of anthelmintics at their m i n i m u m inhibitory concentrations on the growth and development of the fourth stage larvae to adult stages of N. brasiliensis, in vitro: a Laevamisole hydrochloride on day 3; b morantel tartrate on day 3; c amoscanate on day 3; d thiabendazole on day 7 ; e avermectin Bla on day 7 ; f phenothiazine on day 7. (All x 65)

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F

C Fig. 2a-e. Development of worms of N. brasiliensis, in wtro during the course of the screening test: a Fourth stage larvae on day 0; b cast cuticle of L~ after moult to adult stage; c adult ~tages on day 7. (All x 65)

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Discussion

The test's performance at detecting the activity of known anthelmintics indicate its promising potential as a primary screen for the selection of compounds having either broad-spectrum anti-nematode activity or specific activity against gastrointestinal trichostrongyles. Such is evident from the fact that the broadspectrum compounds, e.g. avermectin, laevamisole, morantel tartrate, thiophahate, the isothiocyanates, and the benzimidazoles all showed activity as did the more narrow spectrum compound, phenothiazine. The screen, however, is of no value for detecting the activity of compounds effective only against ascarids, oxyurids, or filarial worms. Compounds of that type, e.g. piperazine, diethylcarbamazine, and suramin were totally ineffective in the screen. Evidence obtained to date from the empirical screening of numerous compounds of diverse structure indicate that the test is highly specific, detecting far less false positive results than in vitro tests against the free-living stages of the parasite. This is an obvious advantage since much time, effort, and money spent developing spurious and irrelevant lead compounds can now be saved. The test has been shown to be economical and simple to operate, requires very little compound, and gives highly reproducible results. Hence many more compounds can be tested than would be possible by conventional in vivo methods. This is the first report of an in vitro test against the parasitic stages of a nematode, capable of detecting the activity of the benzimidazole group of anthelmintic compounds. Its success in this respect probably relates to the fact that the culture medium used supported the growth and development of the worms. Actual growth would seem to be essential since, if adult worms are used in this test, the activity of the benzimidazoles though detectable is far less marked (Jenkins, unpublished work). Coles and McNeillie (1977) failed to detect benzimidazoles and thiophanate in a test using adult N. brasiliensis in M.M.P. saline and we also consistently failed when using several commercially available media enriched with serum and erythrocyte lysate. None of these media supported the growth of the worms. It has been suggested by Coles and McNeillie (1977) that the inactivity of the benzimidazoles in their medium resulted from the inability of the worms to feed in culture and thus concentrate the drug. While this may be true the fact that these compounds were less active against adult than the developing fourth stage larvae in our medium indicates either that fourth stage larvae are more susceptible to benzimidazoles or, more likely, that growth of the worms in culture is an important requirement for their activity to be expressed. There can be no doubt that adult worms feed in our culture medium because they remain active and continue to produce eggs, though infertile, for over three weeks in culture without showing overt signs of deterioration. A problem common to many in vitro screens is the lack of suitable parameters for detecting the activity of compounds objectively. Changes in the degree of motility of worms in culture are extremely difficult to measure objectively as is their rate of growth. Biochemical and physiological parameters, e.g. the rate of excretion of gases, the incorporation of radio-labelled substances, or the

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measurement of net increases in protein, etc. can be measured objectively but require elaborate equipment and are labour intensive. The present test, however, has one wholly objective parameter, namely the occurrence or not of the moult from the fourth larval to the adult stage. The cast cuticles found after moulting has occurred, are easily observed in the test chambers and can be counted rapidly under an inverted microscope at low power. Further, this parameter is exploitable for compounds with very different modes of action, every active anthelmintic tested so far, with the exception of bephenium, showing a significant inhibitory effect on moulting. One hazard common to all in vitro screening tests is their inability to detect the activity of those compounds that require first to be metabolized by the host. However, such a harzard is also common to many in vivo tests, e.g. those in rodents where the target of such screening is a compound for use against the parasites of sheep and cattle. The metabolism of a compound in a rat or mouse bears little resemblance to that in polygastric ungulates. Further, this one disadvantage of in vitro screening is greatly outweighed by the advantages, namely, economy, speed, and the far greater coverage of experimental compounds made possible by the use of an in vitro system. So far the best medium is undefined, employing some variable components such as a tryptic digest of casein and yeast extract. However, during the course of this work many different batches from the same supplier and from different suppliers have been used equally successfully to support the growth and moulting of the worms. Clearly, further studies to refine the medium should be undertaken.

References Coles GC, McNeillie RM (1977) The response of nematodes in vivo and in vitro to some anthelmintics. J Helminthol 51:323-326 Keeling JED, Lewis GA, Jenkins DC (to be published) A modified medium for the in vitro cultivation of trichostrongylid nematodes and its use for anthelmintic screening. Trans R Soc Trop Med Hyg (Abstract) Leland SE, Ridley RK, Slonka GF, Zimmerman GL (1975) Detection of activity for various anthelmintics against in vitro-produced Cooperia punctata. Am J Res Vet Sci 36:449-456 Platzer EG, Eby JE, Friedman P A (1977) Growth inhibition of Caenorhabditis elegans with benzimidazoles. J Nematol 9:280 Tiner JD (1958) A preliminary in vitro test for anthelmintic activity. Exp Parasitol 7:292305 Vanfleteren JR, Roets DE (1972) The influence of some anthelmintic drugs on the population growth of the free-living nematodes, Caenorhabditis briggsae and Turbatatrix aceti (Nematoda: Rhabditoidea). Nematologica 18:325-338

Received May 8, 1980