Controlled Laboratory Trials in Sheep with the Anthelmintic Morantel

Br. vet. J. ( [970), 126, [42

CONTROLLED LABORATORY TRIALS IN SHEEP WITH THE ANTHELMINTIC MORANTEL By R. L.

CORNWELL AND

R. M.

JONES

Therapeutics Research Division, The Pfizer Group, Sandwich, Kent

SUMMARY

The anthelmintic activity of morantel has been demonstrated in controlled laboratory trials involving natural and experimental infections in sheep. Single oral doses of 6 mg. /kg. and higher of a 2 per cent aqueous solution of the . tartrate salt resulted in high activity against Haemonchus, Ostertagia, Trichostrongylus, Nematodirus, Cooperia, Oesophagostomum and Chabertia. Activity against Trichuris and Dictyocaulus was low or absent. Immature stages of abomasal and small intestinal worms were also highly susceptible to the drug. The safety margin is approximately twenty times the therapeutic dose of 10 mg. /kg. INTRODUCTION

The discovery of a new series of compounds active against a wide range of nematodes parasitic in the gastro-intestinal tract of animals was reported by Austin et at. (1966). One of these compounds- pyrantel*-has undergone extensive evaluation and found commercial application in sheep, cattle and horses (Cornwell, 1966; Cornwell et at., 1967; Cornwell & Jones, 1968, I970a ). A further compound in this group, the 3-methyl analogue of pyrantel, given the common name morantel, has proved more potent than pyrantel in mice and dogs (Howes, 1968), and its activity in sheep is reported here.

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Morantel tartrate is a white, odourless, non-staining crystalline solid readily soluble in water (16'3 per cent w./v. at 23 °c) . The fumarate salt has similar • "Banrninth"-"Strongid"- Pfizer Ltd.

TRIALS WITH MORANTEL IN SHEEP

143

physical characteristics with a lower solubility (3.6 per cent w. /v.). Both salts were used in aqueous solution and sheep were treated orally by bottle drench.

Natural infections Sheep were obtained from farms with a history of parasitic gastro-enteritis and allocated to groups on the basis offaecal egg counts. At slaughter, 5 days after treatment, appropriate areas of the intestinal tract were removed separately and the contents and mucosal washings screened with a 100 mesh sieve. The debris remaining on the sieve was suspended in 4 litres of water and after thorough mixing an aliquot of 200 ml. was examined for worms. In experiments 2 and 3 the abomasal mucosa was subjected to digestion in pepsin- hydrochloric acid (Herlich, 1956) for recovery of his totrophic stages, and all worms classified into fifth stage and adults by genus and fourth stage without identification. In experiment I the abomasal mucosa was not digested and fifth stage worms were grouped with adults. Experimental infections Laboratory strains of Haemonchus contortus, Trichostrongylus colubriformis, Nematodirus battus and Dictyocaulus filaria maintained by passage in worm-free sheep were used for the establishment of experimental infections. Animals were assigned at random to the different treatment groups, including controls. At slaughter, 5- 8 days after the last treatment, worms were recovered from 'the gastro-intestinal tract by washing and sieving as described above. D. filaria were recovered from the lungs by opening the bronchial tree with scissors, removing all visible worms and then allowing the remaining worms to free themselves in saline in a Baermann apparatus left overnight in the 37°C incubator. RESULTS

Natural infections-experiments 1-3 The results of three experiments involving a total of fifty-one naturally infected sheep are shown in Table I. In experiment I treatment groups of 12·5 and 25 mg. /kg. morantel fumarate were compared with controls. The principle species present were intestinal Trichostrongylus and Ostertagia. Both dosage levels were equally effective in removing all adult and immature abomasal worms from the lumen. Somewhat lower efficacy 82- 85 per cent was r ecorded against Trichostrongylus spp. in the small intestine, but the light infections of Nematodirus and Cooperia were removed, together with 96-97 per cent of intestinal fourth stage larvae. In the large intestine Chabertia infection was removed by the higher dose and 82 per cent efficacy was recorded for the lower dose. Trichuris was not affected by either dosage. Experiment 2 was a dose-response investigation using the tartrate salt. Dosages compared were 3, 6, 15 and 25 mg. /kg. and the infection was very heavy with most of the common species present in substantial numbers. At dosages of 6 mg. /kg. and higher, almost complete removal of immature and adult abomasal worms was achieved from the lumen and the mucosa. At the

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TABLE I EXPERIMENTS 1-3: ANTHELMINTIC ACTIVITY OF MORANTEL FUMARATE AND TARTRATE SALTS IN NATURAL INFECTIONS

Exp.

I.

Fumarate salt

Exp.

2.

% Activiry at Wonns in controls Range Mean

Species Haemonchus contortus Ostertagia spp.

Adults Fifth stage

80

0-200

12'5

(mg./kg.) 100

Adults Fifth stage

1150

Trichostrongylus axei

Adults Fifth stage

27 0

Unidentified abomasal

Fourth stage

95 0

Trichostrongylus spp.

Adults Fifth stage

43 20

Nematodirus spp.

Adults Firth stage

80

Cooperia spp.

Adults Fifth stage

120

0-2 4 0

100

Unidentified intestinal

Fourth stage

680

3 6 0- 1 4 00

96

Oesophagostomum venulosum

Adults

Trichuris ovis

Adults

'5

0-2 7

Chabertia ovina

Adults

80

0-2 4 0

440-2200 0-7 60

100

100 100

3.

Tartrate salt

% Activiry at Wonns in controls Range Mean

25

Exp.

Tartrate salt

1350 13 8 200 57 4 260

3

6

15

25

(mg./kg.)

80-57 20 0-480

93 64

100 100

100 100

3970-445 20 5 6 0- 14480

85 86

100 99

100 100

Wonns in controls Mean Range

% Activi~y at 7'5

10

(mg./kg.)

533 173

0- 14 80 0-280

100 100

29 2 7 3 80

1080-6880 40-1160

99 100

100 100

100 100

100 100

100 100

100

100

3 611 1117

1570-6590 180-185 0

85 93

99 99

100 100

100 100

1740 20 7

5 6 0-4 120 120-360

100 100

100 100

200-3 120

100

100

49 6 5

1050-12690

87

100

100

100

1960

40-49 60

100

100

2600-56 4 0

82

85

7737 753

4920-11680 200-15 20

23 77

85 100

99 100

100 100

85 80 467

86 100

98 98

100

100

4973 447

1640-10520 200-880

~:

100 100

100 100

100 100

1340 147

0-43 20 0-400

100 100

100 100

100

1827 273

7 6 0-3 8 4 0 80-480

100 100

100 100

100 100

100 100

860 133

200-2200 0-320

100 100

100 100

97

12 53

600-19 60

63

100

100

100

353

o-g60

100

100

98

98

0-280

133

0-280

93

85

100

100

28

26 40- 1 5440 120-880

II-50

0

0

67

4- 111

66

12

25

6,

46

6-129

0

0

88

100

20

0-120

100

50

100

100

29

8-76

91

97

Experiment I four sheep per group. Experiment 2 five sheep per group. Experiment 3 four sheep per treated group and six controls

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TRIALS WITH MORANTEL IN SHEEP

lower dose of 3 mg. /kg. high efficacy was retained against most abomasal species with 64-93 per cent removal. . In the small intestine complete removal of adult and immature Nematodirus and Cooperia and immature Trichostrongylus was achieved at dosages of 6 mg./kg. and higher. The lower dosage of 3 mg. /kg. was still IOO per cent effective against Cooperia and only slightly less so against Nematodirus, but activity against Trichostrongylus adults had dropped to 23 per cent. In the large intestine high activity was present against Oesophagostomum venulosum at all dosages, but the results against Trichuris were difficult to interpret, there being no true dose-response relationship and probably indicates little activity. Chabertia was present in only one control animal and the results are therefore only tentative. Experiment 3 was an attempt to determine the optimum therapeutic dose which was judged from experiment 2 to be somewhere between 6 and 15 mg./kg. In this experiment, therefore, dosages of 7'5 and 10 mg. /kg. tartrate salt were compared with controls. The infection consisted of moderate numbers of most common species. The IO mg. /kg, dosage removed IOO per cent of adult and immature Haemonchus, Ostertagia, T. axei, Nematodirus and Cooperia; 98 per cent of Trichostrongylus spp. and Oesophagostomum and 97 per cent of Chabertia. The lower dose of 7'5 mg./kg. was equally effective except for Trichostrongylus adults and Chabertia when the activity fell to 86 per cent and 91 per cent respectively. Neither dosage level was effective against Trichuris.

Experimental infections Experiments 4 and 5- Trichostrongylus colubriformis (Table II) . In experiment 4 eleven worm-free Dorset Horn lambs were infected with 50,000 T. colubriformis larvae. When the infection was mature-day 28-the lambs were divided into two treatment groups offour lambs each dosed with 13 and 26 mg. /kg. fumarate salt respectiveiy in 5 per cent solution and one group of three untreated lambs as controls. At slaughter 5 days after treatment the controls harboured a uniform infection of 32,600 worms indicating a development of 65 per cent of the inoculum. The treated groups showed a reduction of 88'4 and 90'2 per cent respectively for the 13 and 26 mg.fkg. treatments. TABLE II EXPERIMENTS

Experiment

4

5

4

AND

5:

ANTHELMINTIC ACTIVITY OF MORANTEL FUMARATE AGAINST EXPERIMENTAL T. colubriformis

No. of sheep in group

Dosage (mg .Jkg.)

4 4 3

13 26

3 4 6

Controls

12'5 25'0 Controls

Mean

Worms recovered post mortem Range

Efficacy (%)

3780 3200 32600

0-8160 40-8960 29680-34880

88'4 90'2

37 20 260 34940

1120-7560 40-880 25200-40600

83.6 99'3

BRITISH VETERINARY JOURNAL, 126,3

Experiment 5 was conducted similarly to experiment 4 except that crossbred lambs were used and treatment groups consisted of three lambs dosed with 12·5 mg.Jkg. and four lambs with 25 mg.Jkg. fumarate salt. Six lambs were left untreated as controls. The results showed that an average of 34,940 worms developed in controls-indicating a 70 per cent establishment of infection. The treatment reduced the worm burden by 83.6 and 99·3 per cent respectively for the 12·5 and 25 mg. Jkg. dosage. Experiment 6-H. contortus, T. colubriformis, N. battus and D.filaria (Table III) . Twenty worm-free Dorset Horn lambs were infected with 9000 D. filaria larvae on day a and with 5000 H. contortus, 50,000 T. colubriformis and 9000 N. battus on day II. The lambs were divided into equal groups comprising four treatment and one control. Treatment with 10 mg. Jkg. tartrate salt was given orally on days 14, 18, 25 and 32 of the experiment to the four treatment groups respectively. This corresponded with the same days of the D.filaria infection and with days 3, 7, 14 and 21 of the infection with the gastro-intestinal species. The lambs were slaughtered for worm counts on days 39 and 40. Six lambs died, primarily from the lungworm infection, between days 26 and 36. Two were in the earliest treatment group and one from each of the remainder including controls. The interval between dosing and death was in each individual case sufficient to regard the worm counts as valid for inclusion in the results of the experiment. TABLE III E XPERIMENT 6: ANTHELMINTIC ACTIVITY OF EXPERIMENTAL INFECTIONS OF H. contortus,

SPecies H. conJQrtus

Day of irifection when treated

3 7 14 21 Controls

T. colubrifonnis

3 7 14 21 Controls

N. battus

3 7 14 21 Controls

D· filaria

14 18 25 32

Controls

10 mg./kg.

MORANTEL

T . colubriformis, N. bat/us Worms post mortem Mean Range

Efficacy (%)

40 190 0 0 1430

120-2360

13 0 420 2880 1910 300 50

0-280 80-800 00-93 20 90-4 040 23 12 0-377 60

99'6 98'6 90'4 93'6

0-40

99'5 100 99'5 99'5

10

0-160 80-240

TARTRATE AGAINST AND D. filaria

0 10 10 259 0

0-40 0-40 1400-49 60

209 524 344 62 5 69 6

63-3 19 82- 1093 114-709 155-977 110- 1334

Four sheep per group,

97 87 100 100

7° 25 50

Nil

TRIALS WITH MORANTEL IN SHEEP

The control lambs showed mean worm counts of 1430 H. contortus (29 per cent development); 30,oso T. colubriformis (60 per cent development); 259 0 N. battus (29 per cent development) and 696 D. filaria (23 per cent development). Treatment with 10 mg./kg. morantel tartrate removed 97-100 per cent of 3, 14 and 21-day-old H. contortus and 87 per cent of 7-day-old worms. Efficacy against T. colubriformis was 99'6, 98'6, 90'4 and 93.6 per cent for 3, 7, 14 and 21-day-old worms respectively and 99'S- IOO per cent for the same stages of N. battus. There was no activity against adult D. filaria. The possibility of some activity against immature lungworm could not be entirely discounted but the worm counts were too variable to permit an assessment to be made.

Toxiciry Experiments involving forty-four sheep drenched orally with dosages ranging from 100-300 mg. /kg. moran tel tartrate resulted in the deaths of one animal out of seven dosed at 200 mg. Jkg., and one out of six dosed at 2S0 mg. Jkg. Death occurred within 6 to 9 hours of drenching and following rapidly after symptoms of ataxia and recumbency. No symptoms were noted in other animals. From the recommended dose of 10 mg. Jkg. this gives a safety margin of approximately twenty. DISCUSSION

Morantel tartrate possesses broad-spectrum anthelmintic activity against adult and immature worms causing gastro-enteritis in sheep. Cooperia spp. were the most susceptible being completely removed by doses as low as 3 mg.Jkg. The abomasal species Haemonchus, Ostertagia and T. axei together with Nematodirus were removed to the extent of 99-100 per cent by 6 mg. Jkg. and above in natural infections. The remaining species present in these trials- Oesophagostomum venulosum, Chabertia and Trichostrongylus spp. in the small intestine were removed to the extent of 97-100 per cent by doses of IO mg. Jkg. and higher. Trichuris ovis responded only marginally (61 per cent) to 25 mg. /kg. In experimental infections, with the exception of fourth stage Haemonchus (87 per cent), IO mg. Jkg. moran tel removed over 90 per cent of all stages of Haemonchus, T. colubriformis and N. battus. No activity was apparent against D. filaria adults and the 25-70 per cent activity recorded against various immature stages must be viewed with caution. The fumarate salt of moran tel, although still highly active at doses between 12'S and 26 mg.Jkg., was somewhat less active than the tartrate salt against intestinal Trichostrongylus spp. This reduced activity may be associated with the lower solubility characteristic of the fumarate. The preferred salt is therefore the tartrate, the recommended therapeutic dose 10 mg.Jkg. giving a safety margin of approximately twenty. The safety of the drug and its performance in preventing parasitic gastroenteritis in fattening lambs has been determined in field trials (Cornwell & Jones, 1970 b).

BRITISH VETERINARY JOURNAL, 126, 3 ACKNOWLEDGMENTS

The authors are indebted to Miss S. G. Rees and Messrs B. Bunbury and C. P. Wilkins for able technical assistance . . The Directors of the Pfizer Group are thanked for permission to publish this paper. REFEREN CES

AUSTIN, W. C., CoURTNEY, W., DANILEWICZ, j. C., MORGAN, D. H., CONOVER, L. H., HOWES, H. L., jr., LYNCH, j. E., McFARLAND, j. W., CORNWELL, R. L. & THEODORIDES, V. j. (1966). Nature, Lond., 212, 1273. CORNWELL, R. L. (1966). Vet. Rec., 79, 590. CoRNWELL, R. L., JONES, R. M., BERRY, j., JORDAN, T., MERCER, E. A., PULLEN, D . A. & RILEY, C. j. (1967). Vet . Rec., So, 434. CORNWELL, R. L. & JONES, R. M. (1968) . Vet. Rec., 82, 586. CORNWELL, R. L. & JONES, R. M. ( 1970a) . Br. vet. ]., 126, 134. CORNWELL, R. L. & JONES, R. M. (1970b). Vet. Rec. (in press) . HERLICH, H. (1956). Proc. helminth. Soc. Wash., 23, 102. HOWES, H. L. Jr. (1968) . 42nd Ann. Meet. Am. Soc. Parasit., 38. (Accepted for publication I October 1969)

Epreuves de laboratoires controlees chez Ie D1outon avec l'anthebnintique Morantel (Cornwell et Jones) ResUD1e. L'activite anthelmintique de moran tel a ete demontree par des epreuves de laboratoires controlees chez Ie mouton atteint d'infections naturelles et experimentales. Des doses orales uniques egales ou superieures a 6 mg./kg. d'une solution aqueuse a 2 pour cent du tartrate ont ete tres actives contre Haemonchus, Ostertagia, Trichostrongylus, Nematodirus, Cooperia, Oesophagostomum et Chabertia. 11 y a eu peu ou pas d'activite c~ntre Trichuris et Dictyocaulus. Les stages immatures des vers de I'abomasal et de I'intestin grele ont ete egalement tres suceptibles au medicament. La marge de securite est d'environ vingt fois Ie dose therapeutique de IOmg./ kg. Laboratoriumsversuche mit Kontrollen bei Schafen wt deD1 AnthelD1intikUD1 Morantel (Cornwell undJones) Zusamrnenfassung. Naturliche und experimenteIIe Infektion von Schafen wurden in Laboratoriumsversuchen mit Kontrollen verwendet, urn die anthelmintische Wirkung von Morantel zu zeigen. Orale Einzeldosen von 6 mg./kg. oder mehr von einer 2 prozentigen wassrigen Losung des Tartratsalzes zeigten starke Wirkungen gegen Haemonchus, Ostertagia, Trichostrongylus, Nematodirus, Cooperia, Oesophagostomum und Chabertia . Gegen Trichuris und Dictyocaulus war die Wirkung gering oder Null. Unreife Stadien von Wurmern des Dunndarms oder des Labmagens waren ebenfalls hochst empfindlich gegen das Praparat. Die Sicherheitsgrenze liegt etwa bei 20mal cler therapeutischen Dosis von IOmg.kg. Pruebas controladas de laboratorio efectuadas en ovejas COD rnorantel antihelrnintico (Cornwell y Jones) ReSUD1en. La actividad antihelmintica del morantel ha sido demonstrada en unas pruebas controladas de laboratorio que estudiaron infecciones naturales y experimentales en ovejas. Una sola dosis de 6 mg./kg. 0 una dosis mayor de una soluci6n acuosa de 2 por ciento de la sal de tartrate resuitaron en una gran actividad contra Haemonchus, Ostertagia, Trichostrongylus, Nematodirus, Cooperia, Oesophagostomum y Chabertia. La actividad contra Trichuris y Dictyocaulus fue poca 0 ninguna. Los estados inmaturos de gusanos abomasales y del intestino delgado fueron tambien alta mente susceptibles a la droga. El margen de seguridad es aproximadamente veinte veces la dosis terapeutica de IO mg./kg.