Efficacy of an in-feed formulation of ivermectin against adult worms and somatic larvae of Strongyloides ransomi

veterinary parasitology ELSEVIER

VeterinaryParasitology 65 (1996) 89-97

Efficacy of an in-feed formulation of ivermectin against adult worms and somatic larvae of

Strongyloides ransomi D. Barth ~'* S. Rehbein" J.F.S. Reid b R.A. Barrick c a Merck Research Laboratories, 83101 Rohrdorf-Lauterbach, Germany b Merck Research Laboratories, Clos du Lynx 5, Bruxelles, Belgium e Merck Research Laboratories, Rahway, NJ 07065, USA

Received 14 August 1995; accepted 28 November 1995

Abstract

The efficacy of an in-feed formulation (IVOMEC ® premix) containing 0.6% ivermectin was tested against Strongyloides ransomi in swine. The efficacy of ivermectin against patent infections of S. ransomi when given via the feed at 2 ppm for 7 days (Days 0-7) to provide 100 mcg ivermectin kg -~ body weight day-~ was evaluated in a study with 16 3-month-old male castrated piglets. Seven days prior to treatment each piglet was infected subcutaneously with 2500 infective larvae of S. ransomi. Fecal egg counts were carded out on Days - 7 , 0, 7 and 14, and worm counts on Day 14. Efficacy was 100% in all treated piglets. Two trials involving 40 pregnant gilts were carded out to evaluate the efficacy of ivermectin against the somatic larval stages of S. ransomi when given at a daily dose of 100 mcg kg- 1 body weight for 7 days starting on Days 66, 78, 92 or 103 of pregnancy. The gilts were each experimentally infected with three subcutaneous injections of 250000 infective larvae, with the last infection given between 12 and 30 days prior to commencement of treatment. Gilts were confirmed free of pre-existing intestinal stages of S. ransomi prior to ivermectin treatment. Fecal nematode egg counts were carded out in gilts/sows and piglets subsequently born. The Strongyloides larvae present in sow milk 1, 2 and 7 days post partum were counted. Fourteen days post nature, worm counts were performed in four randomly selected piglets from each litter. IVOMEC ® premix given to pregnant gilts prevented shedding of larvae in sow milk, egg output in feces and the establishment of S. ransomi in piglets. Keywords: Pig; Nematoda; Strongyloides ransomi; Ivermectin;Control methods

* Corresponding author. Tel.: 49-8032-501l; fax: 49-8032-1310. 0304-4017/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0304-401 7(96)00939-9

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1. Introduction Strongyloides ransomi is an extraordinary nematode. Not only is it a common endemic parasite of swine (Aka, 1994), but it may also complete a free-living life cycle. The parasitic generation consists only of parthenogenetic females located in the small intestine. Adult worms are primarily found in suckling piglets whereas sows commonly host somatic larval stages. Infections may be acquired by ingestion, skin penetration and most importantly by transcolostral passage of infective third-stage larvae from the sow to the newly born piglets (Moncol and Batte, 1966). The enteric stages of S. ransomi have been shown to be susceptible to thiabendazole (Leland et al., 1968; Pfeiffer and Supperer, 1969), cambendazole (Egerton et al., 1970; Enigk and Dey-Hazra, 1971) and parenterally administered ivermectin (Barth et al., 1980; Stewart et al., 1981; Brokken et al., 1983; Gill et al., 1990). The somatic third-stage larvae found in the sow are much more refractory to treatment (Moncol and Batte, 1971; Enigk et al., 1974; Barth, 1980). Murrell (1981) demonstrated an efficacy of 87% against arrested L 3 in the subventral fat of fattening pigs when ivermectin was given intramuscularly at 600 mcg kg -~. When given at the recommended single dose of 300 mcg kg- ~ subcutaneously to sows once, 4 to 16 days before farrowing, ivermectin was highly effective against somatic S. ransomi larvae as judged by counts of larvae in milk and the worm burdens of piglets (Barth and Preston, 1985). This paper reports the results of three controlled studies designed to evaluate the efficacy of ivermectin in an in-feed formulation against adult S. ransomi in piglets (Trial 1) and its ability to prevent transmission of somatic larvae from the sow to its progeny (Trials 2 and 3).

2. Materials and methods 2.1. Animals 2.1.1. Trial 1

Sixteen male-castrate crossbred piglets (Deutsches Edelschwein × Pietrain) weighing between 25.6 and 33.6 kg and approximately 3 months old, were used for the study against adult S. ransomi. The animals were born and raised under worm-free conditions. 2.1.2. Trials 2 and 3

Forty pregnant Deutsches Edelschwein (10-12 months old) gilts weighing between 123.2 and 186.0 kg at time of treatment, and from a S. ransomi free herd, were used for the studies against somatic larvae. In both studies the gilts used were inseminated on two different dates. Within each date the gilts were ranked according to body weight before the start of the first treatment period to form replicates of three gilts each. In each trial the gilts of Replicates 1-3 and 4-6 (Trial 3) or 4-7 (Trial 2) were artificially inseminated on the same day.

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2.2. Infection

A recently isolated field strain of S. ransomi originating from Northern Germany and maintained by two (Trial 3) or three (Trials 1 and 2) passages was used. The larvae were cultured from feces of infected donor animals and stored at room temperature until use. At time of infection they were 1 - 3 days old. The piglets were each infected with 2500 larvae of S. ransomi by subcutaneous injection in the inguinal region 7 days prior to treatment. The gilts, were infected with 250000 infective larvae by subcutaneous injection in the neck, on each of three occasions between the 34th and 80th day of gestation (Table 1). After artificial infection all gilts were treated with thiabendazole. At 50 mg kg-1 this compound removes any enteric stages of S. ransomi, which might act as a source of infection to the piglets, however, it has no significant effect against somatic larvae of S. ransomi. This was done in Trial 2 10 days after last infection while in Trial 3 all gilts were treated 7 days after last infection. As some Strongyloides eggs were found in the feces of control gilts of Trial 3 21 days after last infection the control gilts were treated a second time the following day to avoid infection of piglets via larvae derived from their feces. 2.3. Treatment

In Trial 1 eight replicates of two piglets of similar body weight were formed to evaluate efficacy against adult S. ransomi. Within a replicate one animal was allocated randomly to an untreated control group or to treatment with ivermectin (0.6% I V O M E C ® premix, MSD AgVet) included in the complete ration at 2 ppm to provide 100 mcg ivermectin kg-n body weight day -1. Medicated feed was given ad libitum from Day 0 to Day 7. In Trials 2 and 3 replicates of three animals with similar body weight and same insemination day were formed to evaluate efficacy against somatic larvae of S. ransomi. Within replicates, the animals were randomly allocated to an untreated control group or to a treatment group with ivermectin (0.6% IVOMEC ® premix, MSD AgVet) at 100

Table 1 Design of Trials 2 and 3 with gilts to evaluate efficacy of ivermectin against somatic larvae of S. ransomi Trial 2 day Trial day Trial 3 day Trial day Event of pregnancy of pregnancy 34 41 48 58 66-72

0-6

66 73 80 87 92-98 102

0-6 10

78-84

12-18

103-109

11-17

Artificial infection Artificial infection Artificial infection Deworming with thiabendazole Treatment of Group 2 Second deworming of control group with thiabendzole Treatment of Group 3

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mcg kg- l body weight day- ~ in mid pregnancy on Days 66 to 72 or 78 to 84 (Trial 2) and in late pregnancy on Days 92 to 98 or 103 to 109 (Trial 3) of pregnancy (Table 1). Treatments were administered via medicated feed containing 10 ppm ivermectin. Each gilt was fed with a specific amount of medicated feed based on her body weight recorded immediately before start of treatment period. Half of the dose was fed in the morning, the other half in the evening. After consumption of the medicated feed unmedicated feed was given up to 1.1 kg (Trial 2) or 1.25 kg (Trial 3).

2.4. Management To preclude cross infections with S. ransomi between animals, piglets (Trial 1) were housed individually starting 14 days prior to treatment. The gilts in Trials 2 and 3 were transferred to individual pens 8 and 5 days, respectively, before start of ivermectin treatment. The piglets in Trial 1 received a commercial ration for growers ad libitum. The feed for the gilts was a mixture of 50% commercial concentrate plus 50% wheat bran. During the medication period animals were restricted fed twice daily, while the feed was offered ad libitum at other times. Suckling piglets from Trials 2 and 3 were offered unmedicated concentrate feed ad libitum from Day 7 post natum onwards.

2.5. Parasitological investigations Fecal nematode egg counts (EPG) were carried out using a flotation method with saturated sodium chloride solution and a detection limit of 10 eggs g-I feces. Feces of piglets was investigated in Trial 1 7 days prior to treatment, at start and end of the treatment period and 1 week later. In Trials 2 and 3 this was done when piglets were 7 and 14 days old. The feces of gilts was investigated 7 and 28 days post infection and 7 and 14 days post parturition. In Trials 2 and 3 the number of Strongyloides larvae present in 100-ml samples of gilt's milk was also counted. A specially designed milking machine was used to milk the gilts (Visser and Zech, 1985). Milk flow was stimulated by intravenous injection of 10 to 15 IU of oxytocin. The milk samples were centrifuged at 1500 rpm for 5 min. The supernatant was removed and filled up with tap water. This process was repeated until the supernatant was clear and all larvae present in the sediment could be counted microscopically. In Trial 1 all piglets were necropsied 7 days after termination of treatment while in Trials 2 and 3 four piglets from each litter were selected randomly from each litter for necropsy 14 days post natum using a die after ranking the piglets according to body weight within sex. The small intestine of the necropsied animals was removed, opened and the contents washed through a 0.15-mm sieve. The residue was collected and the worms present in two aliquots of 20% each were counted. Geometric means were calculated for worm counts, larval counts and egg counts. The means were compared using Wilcoxon's rank sum statistic and a two-sided significance level of P = 0.05.

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3. Results The results o f Trial 1 showed that ivermectin given in a complete ration at 2 ppm for 7 days was 100% effective ( P < 0.01) against an established infection of adult S. r a n s o m i (Table 2). Feed consumption or weight gain was not influenced by medication o f the feed. During the medication period the treated pigs consumed 54.5 g k g - 1 body weight daily while the controls ate 52.9 g, calculated on body weight at D a y 7. The mean daily weight gain per pig during the treatment period was 713 g in the control group and 750 g in the treated group. In Trial 2 S. ransomi eggs were recovered from feces o f all gilts 7 days post last infection, while after treatment with thiabendazole no animal excreted eggs. Larvae o f S. r a n s o m i were present in the milk of all control animals with the highest mean numbers observed on the first day and lowest numbers 7 days post partum. N o larvae were recovered from the milk o f animals treated with ivermectin for 7 days commencing 48 to 49 days (Group 2) or 34 to 39 days (Group 3) before parturition. At necropsy 14 days after parturition all control piglets were infested with a mean burden o f 632 S. ransomi and a range of 195-2003 worms. N o worms were recovered from the litters o f gilts treated with ivermectin (Table 3). After assignment, two animals were removed from the control group, one which aborted on Day 4 (Day 70 of pregnancy) and another which

Table 2 Trial 1. Efficacy of ivermectin vs adult S. ransomi in piglets when given orally at a rate of 2 ppm in complete ration for 7 days Control untreated Number of piglets EPG a Day - 7 Day 0 Day 7 Day 14 Efficacy (%) Worm count Mean a Range Efficacy (%) Body weight c (kg) Day - I Day 7 Feed consumption e (g) Days 0-7

lvermectin, 2 ppm

8

8

0 30.4 280.9 87.8 -

0 36.1 0b 0b 100

1105.3 650-1400 -

0 0 100

29.2 34.9 12931

28.9 b 34.9 b 13229 b

a Geometric mean based on transformation In(count + 1). b Based on seven pigs (one pig died on Day 3 due to verrucose endocarditis). c Arithmetic mean.

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Table 3 Trial 2. Efficacy of ivermectin against somatic larvae of S. ransomi in gilts when given orally at a daily dose of 100 mcg kg- ~ body weight for 7 days during mid pregnancy

Number of gilts with litter Duration of pregnancy (days) Number a of piglets per litter Bona At 14 days Weight a (g) of piglets At birth At 14 days E I ~ e of gilts 7 days post infection 28 days post infection 7 days post parturition 14 days post parturition E I ~ ¢ of piglets At 7 days At 14 days Larvae counts c lOOml- l milk 1 day post partum 2 days post partum 7 days post partum Count c of S. ransomi per piglet At 14 days Efficacy (%)

Control untreated

lvermeetin during gestation days 66-72

78-84

5 113-116

7 114-115

7 112-117

10.2 7.60

10.7 8.00

8.4 6.00

1147 3573

1211 3590

1213 3249 b

l 145 0 0 0

1334 0 0 0

957 0 0 0

81 l 5057

0 0 b

23.0 16.1 0.9 632 d

0 0 0b 0

0 d

100

100

a Arithmetic mean. b Counts from four piglets of four sows each and two piglets of one sow. c Geometric mean based on transformation In(count+ 1). a Based on six sows (one sow had no milk and piglets died within 1 day).

did not b e c o m e pregnant. In G r o u p 3 one animal was v e r y a g g r e s s i v e and killed its piglets within 1 day post partum. In Trial 3 S. r a n s o m i e g g s w e r e present in feces o f all gilts 7 days p o s t infection but not after treatment with thiabendazole. W h i l e Strongyloides larvae w e r e present in milk o f all control gilts n o larvae w e r e seen in the m i l k o f gilts treated with i v e r m e c t i n for 7 days starting 2 0 - 2 4 days ( G r o u p 2) or 8 - 1 3 ( G r o u p 3) days b e f o r e farrowing. The m e a n w o r m burden o f control piglets necropsied at an age o f 14 days w a s 553 S. ransomi with a range o f 1 1 3 - 1 6 3 3 w o r m s . E f f i c a c y was > 9 9 % ( P < 0.01) for those litters f r o m gilts treated f r o m D a y 92 to 98 o f gestation and 100% for those treated f r o m D a y 103 to 109 (Table 4). In Trial 3 one control gilt did not a l i o w its piglets to suckle and all piglets died within 4 days.

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Table 4 Trial 3. Efficacy of ivermectin against somatic larvae of S. ransomi in gilts when given orally at a daily dose of 100 mcg k g - i body weight for 7 days during late pregnancy

Number of gilts with litter Duration of pregnancy (days) Number a of piglets per litter Born At 14 days Weight a (g) of piglets At birth At 14 days EPG ¢ of gilts 7 days post Infection 28 days post infection 7 days post parturition 14 days post parturition EPG c of piglets At 7 days At 14 days Larvae counts b 100ml- ~ milk 1 day post partum 2 days post partum 7 days post partum Count c of S. ransomi per piglet At 14 days Efficacy (%)

Control untreated

lvermectin during gestation days 92-98

103-109

6 112-116

6 112-116

7 111-116

8.33 6.33 b

8.17 7.67

11.14 9.57

1423 3501 b

1354 3827

1260 3318

3842 0 0 2

3191 0 0 0

2238 0 0 0

153 b 1720 b 8.8 5.7 2.2 b 553 b -

0.1 d 0.1 a 0 0 0 0.2 d > 99

0 100

a Arithmetic mean. b Based on five sows. One sow removed from trial 4 days post partum, after all her piglets died. c Geometric mean based on transformation In(count + 1). d A single piglet.

In none of the trials an adverse reaction attributable to IVOMEC ® premix was observed.

4. Discussion

Our results show that ivermectin given orally at 2 ppm in a complete ration over a period of 7 days was highly effective (100% efficacy) against adult S. ransomi. This level of efficacy is equivalent to that provided by a single injection of 300 mcg ivermectin kg -~ body weight (Benz et al., 1989; Gill et al., 1990; Xie et al., 1991). Somatic larvae do not continue their development until they are transmitted by the mother to her offspring. This was confirmed by the fecal examinations of the gilts which showed that after successful treatment of the enteric infection with thiabendazole gilts did not shed Strongyloides eggs throughout until the trial was terminated 14 days post

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parturition. As fecal excretion in the sows was controlled the only source of infection in piglets could have been the somatic larvae in the colostrum. While a number of anthelmintics are available for treatment of patent infections of S. ransorai, ivermectin is the only compound which has also been shown to be effective against the somatic larvae of this parasite. The results show that transfer of S. ransomi larvae to suckling pigs via milk or colostrum was prevented when sows were treated with oral ivermectin at 100 mcg kg-~ day-~ for 7 consecutive days during pregnancy. The studies reported here also confirmed that the high efficacy of oral ivermectin against somatic larvae in the pregnant sow is similar to that seen after a single injection at a dose rate of 300 mcg ivermectin kg- ] body weight (Barth and Preston, 1985). Treatment was equally efficacious whether given in mid or late pregnancy. Consequently the timing of treatment of S. ransomi, using a convenient in-feed formulation of ivermectin can be flexible. It allows herd treatment to commence on any day during mid to late pregnancy and other factors can be taken into account such as the best timing of treatments for other parasites which the farmer might want to control. References Aka, E., 1994. Epidemiologische Untersuchungen zum Vorkommen yon Endoparasiten in Schweinezuchtbetrieben des Weser-Ems-Gebietes unter besonderer Berficksichtigung yon Strongyloides ransomi und dessen Behandlung mit lvermeetin (IVOMEC-S®). Diss. Vet. Med., Hannover. Barth, D., 1980. Zur Chemoprophylaxe der galaktogenen Strongyloidesinfektiou beim Schwein mit Cambendazol. Prakt. Tierarzt, 61: 119. Barlh, D. and Preston, J.M., 1985. Efficacy of ivermectin against somatic Strongyloides ransomi larvae. Vet. Rec., 116: 366-367. Barth, D., Sutherland, I.H., Roncalli, R.A. and Leaning, W.D., 1980. The efficacy of ivermectin as an antiparasitic agent in the pig. In: H.C. Nielsen, P. Hogh and N. Bille (Editors), Proc. 6th IPVS Congr., 30 June-3 July 1980, Copenhagen, Denmark. International Pig Veterinary Society, p. 275. Benz, G.W., Roncalli, R.A. and Gross, S.J., 1989. Use of ivermectin in cattle, sheep, goats and swine. In: W.C. Campbell (Editor), lvermectin and Abamectin. Springer, New York, pp. 215-229. Brokken, E.S., Barth, D., Foster, A.G., Pulliam, J.D. and Wallace, D.H., 1983. Ivermectin: a new broad-spectrum antiparasitic agent for swine. In: C. Main (Editor), Proc. Merck Symposium: Recent Developments in the Control of Animal Parasites, XXil World Vet. Congr., 25-26 August, Perth, Australia. World Veterinary Congress Organizing Committee, pp. 35-36. Egerton, J.R., DiNetta, J., Neu, D.C., Walter, R.J. and Campbell, W.C., 1970. The efficacy of cambendazole against Ascaris suum and Strongyloides ransomi in swine. Res. Vet. Sci., 11: 590-592. Enigk, K. and Dey-Hazra, A., 1971. Zur Therapie des Strongyloidesbefalls beim Schwein. Dtsch. Tier~rztl. Wschr., 78: 419-422. Enigk, K., Weingkrtner, E. and Schmelzle, M., 1974. Zur Chemoprophylaxe der galaktogenen StrongyloidesInfektion beim Schwein. Zbl. Vet. Med. B, 21: 413-425. Gill, B.S., Singh, J., Singh, R.A.G.B. and Singh, K.S., 1990. Efficacy of ivermectin against mange and gastrointestinal nematodes of pigs and goats. Indian J. Parasit., 14: 93-98. Leland, S.E., Jr., Combs, G.E. and Wallace, L.J., 1968. Anthelmintic activity of thiabendazole and trichiorphon gainst migrating and adult Strongyloides ransomi in suckling and weanling pigs. Am. J. Vet. Res,, 29: 797-806. Moncoi, D.J. and Batte, E.G., 1966. Transcolostral infection of new born pigs with Strongyloides ransomi. Vet. Med./Small Anita. Clin., 61: 583-586. Moncol, D.J. and Batte, E.G., 1971. Porcine Strongyioides: integration of life cycle and therapeutics. In: J. Velazquez (Editor), Proc. 19th World Vet. Congr. Vol. 1. World Veterinary Congress Organizing Committee, pp. 91-95.

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Murreil, K.D., 1981. Induction of protective immunity to Strongyloides ransomi in pigs, Am. J. Vet. Res., 42: 1915-1919. Pfeiffer, H. and Supperer, R., 1969. 0ber die Bekhmpfung der Strongyloidose der Sangferkel mit Thiabendazole. Wien. Tierhrzti. Wschr., 56: 116-120. Stewart, T.B., Marti, O.G. and Hale, O.M., 1981. Efficacy of ivermectin against five genera of swine nematodes and the hog louse, Haematopinus suis. Am. J. Vet. Res., 42: 1427-1428. Visser, M. and Zech, V., 1985. Zur maschinellen Gewinnung yon Sanenmilchproben. Prakt. Tierarzt, 66: 380. Xie, M.Q., Zhang, J.F., Zhang, F.Q., Wu, H.X. and Wen, L.N., 1991. Experiment on the ability of ivermectin to remove parasitic nematodes from the intestines of pigs. Chin. J. Vet. Med., 17: 27-28.