Parasitol Res (2004) 93: 419–422 DOI 10.1007/s00436-004-1149-x

ORIGINAL PAPER

A. I. Njue Æ R. K. Prichard Efficacy of in calves against a resistant oncophora field isolate

Received: 24 February 2004 / Accepted: 28 May 2004 / Published online: 26 June 2004 Springer-Verlag 2004

Abstract Since 1999, two Cooperia oncophora isolates, injectable formulations have been shown to have lower originally obtained from the United Kingdom, have than expected efficacy against this species (Benz et al. been maintained by regular passage through calves at 1989; Bisset et al. 1990; West et al. 1994; McKenna the Macdonald Campus, McGill University farm. One 1995). C. oncophora is the dose-limiting species for isolate, IVS, was originally susceptible to ivermectin, ivermectin, and the lower efficacy against this species while the IVR isolate was originally resistant to iver- may exert a high selection pressure for resistance (Ver- mectin. These two isolates have been used to study the munt et al. 1995). mechanisms of ivermectin resistance. To confirm the Cooperia species resistant to ivermectin have been susceptible/resistant status after 4 years of passaging identified under field conditions in New Zealand (Ver- through calves, a controlled study was performed in munt et al. 1995, 1996; Familton et al. 2001), the United which two worm-free calves were experimentally in- Kingdom (Coles et al. 1998, 2001), and Argentina (An- fected with IVS and another two worm-free calves with ziani et al. 2001; Fiel et al. 2001). Our work focuses on the IVR infective larvae. The calves were treated with understanding the mechanisms of ivermectin resistance ivermectin (0.2 mg/kg) subcutaneously (Ivomec Injec- in C. oncophora. An ivermectin-resistant field isolate tion) 21 days after infection. Ivermectin at the recom- (IVR), originally identified by Coles et al. (1998), has mended dose was 100% effective at eliminating the IVS been central to our work. A second isolate, designated as isolate, since no eggs were found in feces, and no adult ivermectin-susceptible (IVS), has also been used. The worms were found in the small intestine of the treated parasites have been maintained at the Macdonald IVS-infected calf. In contrast, the IVR-infected calf Campus Farm by regular passage through male Holstein continued to pass eggs in feces even after treatment with calves. To establish the susceptible/resistant status of the ivermectin, and adult worms (250) were found in the two isolates after 4 years of passaging, they were sub- small intestine at necropsy. The untreated calves had jected to a controlled efficacy test. 1,330 and 848 adult worms, respectively, for the IVS and IVR infected . Materials and methods

Parasites Introduction Two C. oncophora isolates (IVS and IVR) were kindly Ivermectin is a highly potent broad-spectrum anthel- provided by Dr. G.C. Coles (University of Bristol, UK). mintic that is widely used in cattle. It is available in The IVS isolate had been maintained without anthel- injectable, oral and topical formulations for use in cattle mintic pressure at Weybridge Experimental Station, (Vermunt et al. 1995). Cooperia oncophora is one of the UK, while IVR represents a field isolate originally ob- target species for ivermectin, though the topical and tained from a farm in Somerset, UK, where ivermectin resistance was reported (Coles et al. 1998). A. I. Njue Æ R. K. Prichard (&) Institute of Parasitology, McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Animals Quebec, H9X 3V9, Canada E-mail: [email protected] Tel.: +1-514-3987729 Four 3-month old calves (117–205 kg body weight) were Fax: +1-514-3987857 used in the study, conducted in July and August 2002. 420

They were housed indoors in individual pens at the were emptied into a sampling bucket, and the small Macdonald Campus, McGill University Farm. The intestine split open longitudinally using scissors. The animals were fed hay and given water ad libitum, along mucosal surface was washed thoroughly with warm with a restricted grain supplement. At 14 days prior to water (38C). One liter aliquots were then processed for the start of the experiment, the calves were drenched worm recovery and enumeration by a modification of with fenbendazole at a dose rate of 10 mg/kg body the agar gel technique of Slotved et al. (1996). A 2% weight to clear any existing helminth infections. Prior to agar solution (Bacto-Agar) was prepared by mixing the experimental infection with IVS or IVR infective larvae, agar with tap water. This was heated to boiling point animals were checked for general health, and feces col- and cooled to 45C. One liter of intestinal washings was lected to test the worm-free status of the animals. It was mixed with 1 l of the 2% agar solution at 45C. The established that the calves were not passing any eggs mixture was then poured into eight trays in which dis- after the fenbendazole treatment. posable J-cloths (22·28 cm) had been placed. The agar gel solidified after a few minutes, and the cloths with the adhering gel were clipped to a stick and placed in a Fecal egg count reduction test plastic container, with the sticks being used to suspend the cloths in the container. The container was then filled To determine the efficacy of ivermectin against IVS and with RPMI medium at 38C, and incubated at 38C for IVR worms, two calves were infected with 10,000 IVS 3 h. The worms migrated out of the gel and collected at infective larvae (L3), and two others were infected with the bottom of the container. These were picked and 10,000 IVR L3 by oral gavage. In each group, one ani- counted. Worms remaining in the agar gel were also mal was treated subcutaneously, on day 21 post-infec- picked and included in the total count. Percent reduction tion, with ivermectin (Ivomec Injection, Merial) at the in adult worm burden was calculated using the formula: recommended dose rate of 200 lg/kg body weight in (CwTw)/Cw·100=%efficacy, where Cw and Tw are cattle, while the second was left untreated to act the worm counts of the control (untreated) and treated as a control. The ivermectin dose was calculated based animals, respectively, at necropsy. on the weight of the animal. Treatments were adminis- tered as subcutaneous injections. Fecal samples were collected per rectum from all Results animals at the time of treatment and 7 and 14 days post- treatment. Egg counts were performed on fecal samples Calves infected with the IVS isolate were in good body using a modified McMaster technique (Ministry of condition, and did not show any clinical signs of para- Agriculture Fisheries and Food 1986). Feces (4.5 g) were sitic gastroenteritis (PGE). Following the treatment of thoroughly mixed in 40.5 ml of water. The suspension one of the IVS-infected animals with ivermectin, no eggs was sieved through a piece of gauze. The strained fluid were observed in the feces (Table 1). In contrast, the two was poured into a centrifuge tube, and centrifuged for calves infected with the IVR isolate had diarrhea, rough 2 min at 1,500 rpm. The supernatant was discarded, and hair coats, and were unthrifty. Even after one of the the sediment loosened by stirring. The tube was then IVR-infected calves was treated with ivermectin, it filled with saturated sodium chloride solution to the continued to pass eggs in the feces, (Table 1), and re- same level as before. The tube contents were thoroughly mained in poor condition. Treatment with the recom- mixed by inverting the tube five or six times, and a mended dose of ivermectin (200 lg/kg body weight) was McMaster slide chamber filled using a Pasteur pipette. 100% effective against the IVS isolate, since no eggs After mixing again, the second chamber on the slide was were seen in the treated IVS animal on days 7 and 14 filled. All eggs in both chambers were counted, and the post-treatment. For the IVR isolate, ivermectin reduced total number multiplied by 10 and expressed as eggs per the fecal egg counts by 76.9% and 77.8% at days 7 and gram of feces. 14 post-treatment, respectively. The efficacy was calculated based on the Table 2 shows adult worm counts for the four calves. percent reduction in fecal egg counts using the formula: Adult worms were found in untreated IVS- and IVR- infected animals. No adult worms were found in the ðÞCe Te =Ce 100 ¼ %efficacy; where Ce and Te are the fecal egg counts of the control Table 1 Pre- and post-treatment fecal egg counts (epg) from (untreated) and treated animals, respectively. Ivermectin untreated calves and calves treated with injectable ivermectin resistance was defined as a less than 95% reduction in Animal Day 0 Day 7 Day 14 fecal egg counts post-treatment (Coles et al. 1992). IVS treated 170 0 0 IVS untreated 110 110 90 Calculating the reduction in worm burden % Reduction – 100 100 IVR treated 190 30 40 Necropsy of all four animals was performed on day 22 IVR untreated 180 130 180 % Reduction – 76.9 77.8 or 23 post-treatment. The contents of the small intestine 421 small intestine of the IVS-infected animals following IVR-infected animal remained in poor body condition treatment with ivermectin. In contrast, ivermectin re- even after treatment, though the egg count was low (40 duced the worm burden of the IVR-infected animal by eggs per gram on day 14 post-treatment). At slaughter, only 70.5%. adult worms were found in the small intestine of this animal, suggesting that ivermectin was ineffective at eliminating the adult IVR worms. Discussion and were found to be inef- fective against ivermectin resistant C. oncophora (Ver- The present study compared the efficacy of ivermectin munt et al. 1995, 1996; Anziani et al. 2001), which against IVS and IVR isolates of C. oncophora, and suggests that ivermectin resistant C. oncophora show confirmed the ivermectin-resistant status of the IVR side resistance to doramectin and moxidectin, both isolate, which was previously reported by Coles et al. members of the macrocyclic lactone class of anthelmin- (1998). While ivermectin was 100% effective against the tics. , fenbendazole and oxfendazole were susceptible IVS isolate, efficacy by the fecal egg count used successfully against ivermectin-resistant C. onco- reduction test (FECRT) against the IVR isolate was phora (Anziani et al. 2001; Coles et al. 1998, 2001). 77.8% on day 14 post-treatment. FECRT results below Levamisole belongs to the imidothiazole class of an- 95% are indicative of anthelmintic resistance (Coles thelmintics, while fenbendazole and oxfendazole are et al. 1992). These findings were supported by the adult . These act via mechanisms worm counts, where treatment of IVR-infected animals that are distinct from those of the macrocyclic lactones. resulted in a 70.5% reduction in adult worms, while no The imidothiazoles act on worm acetylcholine receptors worms were found in the treated IVS-infected animal. (Martin et al. 1997), while the benzimidazoles act on The untreated IVS-infected animal had a higher tubulin (Lacey and Prichard 1986). Chloride channels worm burden than the IVR-infected animals, yet the gated by the neurotransmitters glutamate and gamma- IVS-infected animal did not show any clinical signs of aminobutyric acid have been identified as targets for the PGE. As stated by Coles et al. (2001), ivermectin-sus- macrocyclic lactones (Holden-Dye and Walker 1990; ceptible isolates appear to be of low pathogenicity Cully et al. 1994; Arena et al. 1995; Martin 1996; Dent compared to ivermectin-resistant isolates. Calves in- et al. 2000; Feng et al. 2002; Njue et al. 2004). fected with 10,000–20,000 ivermectin-susceptible L3 did not show any clinical signs of disease (Borgsteede and Acknowledgements The authors are very grateful to Dr G.C. Coles Hendriks 1979; Coop et al. 1979), while animals infected for providing the Cooperia oncophora isolates. We also thank G. Bingham and D. Eshelby for technical assistance. This study with 15,000 or 20,000 ivermectin-resistant L3 showed was funded by Fort Dodge Animal Health and NSERC, Canada. edema, weight loss and diarrhea (Coles et al. 2001; The experiments reported here comply with the current laws of Anziani et al. 2001). Cooperia is generally considered a Canada parasite of low pathogenicity, and young calves develop immunity to this parasite following exposure in the first grazing season (Coop et al. 1979; Vermunt et al. 1995; References Claerebout et al. 1999). However, at high infection levels (a single dose of 200,000 L3, or 10 000 larvae per day for Anziani OS, Zimmermann G, Guglielmone AA, Vazquez R, Suarez 6 weeks), Cooperia can cause clinical disease (Borgsteede V (2001) Avermectin resistance in Cooperia pectinata in cattle in Argentina. Vet Rec 149:58–59 and Hendriks 1979; Armour et al. 1987). 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