Reduced efficacy of moxidectin and abamectin in young red deer (Cervus elaphus) after 20 years of moxidectin pour-on use on a New Zealand deer farm

Veterinary Parasitology 199 (2014) 81–92

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Reduced efficacy of moxidectin and abamectin in young red deer (Cervus elaphus) after 20 years of moxidectin pour-on use on a New Zealand deer farm C.G. Mackintosh a,∗ , C. Cowie a , K. Fraser b , P. Johnstone a , P.C. Mason c a b c

AgResearch Invermay, PO Box 50034, Mosgiel 9053, New Zealand AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North 4442, New Zealand Consultant Parasitologist, 317 Dunns Crossing Road, RD 8, Christchurch, New Zealand

a r t i c l e

i n f o

Article history: Received 28 June 2013 Received in revised form 16 September 2013 Accepted 20 September 2013 Keywords: Red deer Moxidectin Abamectin Efficacy Pharmacokinetics Anthelmintic resistance FECRT

a b s t r a c t A study was undertaken on weaned 4–5 month old farmed red deer to test the efficacy of moxidectin and abamectin anthelmintics, given by three different routes of administration, compared with an untreated control. Faecal samples were collected on days 0, 7 and 14 for a faecal egg count reduction test (FECRT), blood samples were collected on days 0, 0.5, 1, 2, 3, 5, 7, 10 and 14 for pharmacokinetics, and the deer were killed on days 14 or 15 for total nematode count. The control group averaged 1264 adult Ostertagia-type nematode parasite species and treatment efficacy was 77.4% for moxidectin injection, 26% for oral moxidectin and 27.6% for pour-on moxidectin, while the treatment efficacy was 72.4% for abamectin injection, 70.1% for oral abamectin (Hi-Mineral) and 34.1% for pour-on abamectin. Both moxidectin and abamectin injections were significantly more efficacious than their equivalent pourons. There was a significant difference in efficacy between oral abamectin (Hi-Mineral) and oral moxidectin (P < 0.01). The control group averaged 2956 adult lungworm (Dictyocaulus eckerti) and 50 Oesophagostomum venulosum in the large intestine and treatment efficacy against these nematodes was 100% for all treatments. There were negligible numbers of other gastrointestinal nematodes. At slaughter, there was a significant correlation (P = 0.02) between FEC and Ostertagiatype nematodes in the untreated controls. Relatively few eggs were found in faeces from treated animals at 7 and 14 days post-treatment despite significant worm burdens in all six treatment groups, suggesting egg-laying suppression in resistant nematodes, and all three different FECRT calculations tended to overestimate the efficacy of the treatments compared with actual nematode counts. Peak plasma concentrations (Cmax ) for both actives were measured 12 h after treatment for injection and oral and at 5 days for pour-on. Cmax (ng/ml) for moxidectin injection, oral and pour-on were 71.8, 8.3 and 0.4, respectively, and for abamectin injection, oral and pour-on were 62.1, 30.3 and 10.0, respectively. Area under the curve (AUC) estimates for moxidectin injection, oral and pour-on were 106.6, 12.9 and 6.1, respectively, and for abamectin injection, oral and pour-on were 162.7, 57.5 and 74.3, respectively. The results demonstrate that significant anthelmintic resistance to moxidectin and abamectin is present on this deer farm. However, the injection was the most effective route of administration in young deer for both anthelmintics, although <80% efficacious. We conclude that the FECRT is unreliable in deer when anthelmintic resistance is present. © 2013 Elsevier B.V. All rights reserved.

∗ Corresponding author. Tel.: +64 3489 9229; fax: +64 3489 9038. E-mail address: [email protected] (C.G. Mackintosh). 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.09.028

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1. Introduction Red deer (Cervus elaphus) have been farmed in New Zealand for over 35 years and the number of breeding hinds in 2011 was estimated to be over 1 million (Statistics New Zealand; http://www.stats.govt.nz). Gastro-intestinal (GI) nematodes and lungworms are recognised as key production limiting factors for farmed deer, especially in their first year of life, and severe parasitism can be fatal (Mackintosh and Wilson, 2003). The strategic use of effective anthelmintics has been essential for the control of GI parasites on deer farms. In the late 1970s and early 1980s, when deer farming first started, lungworms, which were originally identified as Dictyocaulus viviparus and subsequently identified as Dictyocaulus eckerti (Johnson et al., 2001), were considered the main nematode problem (Mason, 1985) and the only anthelmintics available were oral benzimidazoles and levamisole. This latter drench was found to be ineffective against lungworm in deer (Mason and Beatson, 1985), while benzimidazole drenches (oxfendazole, fenbendazole and albendazole) appeared to have moderate efficacy, at least against adult lungworms (Mackintosh et al., 1984). In the early 1980s ivermectin (Ivomec), a member of a new family of anthelmintics, the macrocyclic lactones (ML) or “mectins”, was registered for use in sheep and cattle in New Zealand and its efficacy in farmed deer was investigated. A slaughter study in red deer showed injectible ivermectin at 200 ␮g/kg was 100% effective against mature and immature lungworm, compared with an oral benzimidazole, febantel at 7.5 mg/kg, which was 99.8% effective against adult lungworm and 85% effective against immature lungworm (Mackintosh and Mason, 1985). In the late 1980s and early 1990s a number of “pour-on” ML anthelmintics became available. Originally marketed for topical application in cattle, they were quickly adopted for farmed deer. Ivermectin (Ivomec Pour-on), and moxidectin (Cydectin Pour-on), which is a closely related milbemycin, were among the first to be licensed for use in deer, while eprinomectin (Eprinex Pour-on) and abamectin (Genesis Pour-on) followed. Moxidectin Pour-on at 0.5 mg/kg was shown to be >99% effective in deer against mature and immature GI nematodes and lungworm (Mackintosh et al., 1993; Waldrup et al., 1998) and had 6 weeks persistent activity (Mackintosh et al., 1997). Over the last 20 years pour-on anthelmintics have become very popular in the deer industry because of the ease of application and perceived efficacy. A survey in 2004/2005 found that moxidectin was the anthelmintic most commonly used (46–58%, depending on class of animal), followed by abamectin, eprinomectin, oxfendazole, ivermectin, albendazole, levamisole and doramectin (Castillo-Alcala et al., 2007). In the last 10 years there have been indications that some resistance to ML in parasites of farmed deer has been developing, as has also been seen in the sheep and cattle industries in New Zealand (Leathwick et al., 2001, 2009). In the 2004/2005 survey of deer farms, 2% of farmers said they had evidence of anthelmintic resistance in their deer, but did not provide further information (Castillo-Alcala et al., 2007). In 2005 a slaughter trial of oral ivermectin,

pour-on ivermectin and pour-on moxidectin showed efficacy of >99.8% against Dictyocaulus and Oesophagostomum spp. However, pour-on moxidectin had suboptimal (94%) efficacy and oral and pour-on ivermectin had poor efficacy against abomasal parasites (Hoskin et al., 2005). This suggests that the efficacy of all three anthelmintics, especially ivermectin, had declined on that property compared with the efficacy demonstrated in studies in the 1980s and 1990s. A recent study demonstrated reduced efficacy of anthelmintics in deer, especially of pour-ons, and showed that injectible moxidectin had higher efficacy than pouron moxidectin against Ostertagia-type nematodes in deer (Lawrence, 2011). Pour-on moxidectin has been used almost exclusively on the AgResearch Invermay deer farm for almost 20 years. Recently, positive faecal egg counts (FEC) in some deer three weeks after treatment indicated that anthelmintic resistance was developing on this farm. Consequently a slaughter study was undertaken in young red deer on this farm to test the efficacy and pharmacokinetics of moxidectin and abamectin, which are the most commonly used ML in deer. 2. Materials and methods 2.1. Experimental design and animals Thirty-five 3–4 month-old red deer calves from the Invermay deer farm were weaned on 27 February and given oral oxfendazole at 4.5 mg/kg (1 ml/20 kg Oxfen C, Merial NZ Ltd). They were vaccinated with Yersiniavax (MSD Animal Health) and given a second dose of oxfendazole two weeks later (12 March, 5 weeks prior to study starting) and then run at pasture to acquire new worm burdens by natural exposure. On 27 March (Day −21) all 35 weaners were given an oral dose of 3.5 ml containing ∼800 L3 mixed GI parasite larvae cultured from faecal samples taken the previous spring from Invermay rising yearling deer. The 35 weaners were grazed together at pasture until April 10 (Day −8) when they were yarded, weighed, faecal sampled for FEC and FLC to monitor burdens, vaccinated a second time with Yersiniavax, drafted into indoor pens and fed on lucerne hay for a week prior to the start of the anthelmintic study to familiarise them with handling and prevent further acquisition of parasites. They were randomly allocated to 7 treatment groups of 5 animals. This study was carried out with the approval of, and in accordance with requirements of the Invermay Animal Ethics Committee, approval number AEC12600. 2.2. Treatment protocol On Day 0 the individual deer were weighed, the groups were randomly allocated to treatment and they received the following treatments at manufacturers’ recommended dose rates: (a) Untreated Control (C). (b) Moxidectin oral (MO): Cydectin Oral Drench for Sheep, Zoetis (formerly Pfizer Animal Health), 1 ml/5 kg (0.2 mg/kg).

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(c) Moxidectin injection (MI): Cydectin Injection for Cattle and Sheep, Zoetis, 1 ml/50 kg (0.2 mg/kg). (d) Moxidectin pour-on (MP): Cydectin Pour-On for Cattle and Deer, Zoetis, 1 ml/10 kg (0.5 mg/kg). (e) Abamectin oral (AO): Genesis Hi-Mineral For Sheep, Merial NZ Ltd, 1 ml/5 kg (0.2 mg/kg). (f) Abamectin injection (AI): Genesis Injection, Merial NZ Ltd, 1 ml/50 kg (0.2 mg/kg). (g) Abamectin pour-on (AP): Genesis Pour-On for Cattle and Deer, Merial NZ Ltd, 1 ml/20 kg (0.5 mg/kg).

The aliquots were washed over a 38 ␮m aperture sieve prior to examination. The nematodes were counted by genus and sex of adults. Approximately 100 male Ostertagia-type nematodes from each animal were identified to species. Trichostrongylus and Oesophagostomum were further identified to species. The lungworm were assumed to be D. eckerti (Johnson et al., 2001). Early 4th stage larvae (EL4) from the abomasum incubations were counted using a counting chamber examined under a compound microscope.

The groups were strategically placed in pens so that the abamectin groups were in one corridor, the moxidectin groups were in another corridor and the Control group was in a third area. Whenever the weaners were brought to the handling pen the Control group was always handled first with a separate set of overalls and gloves, and the moxidectin groups and the abamectin groups were also handled with separate overalls and gloves and the oral, injectible and pour-on groups were always handled in that order.

2.3.3. Lungworm counts The lungs were removed at slaughter and the visible airways opened up with fine scissors and all visible lungworms were collected and a 10% aliquot counted. The lungs were soaked overnight in warm saline, agitated and the washings examined for immature lungworm using a 10% aliquot.

2.3. Treatment and sampling On Day −1 the deer were all weighed. On Day 0 the deer were all faecal sampled for FEC and FLC, a 10 ml heparinised blood sample was taken by jugular venipuncture and they were treated strictly according to liveweight with the volume rounded to the nearest 0.1 ml using 1, 3, 5 or 10 ml syringes and the time of treatment noted. Further heparinised blood samples were taken 12 h after treatment and then on Days 1, 2, 3, 5, 7, 10 and 14 to measure the changes in plasma levels of moxidectin and abamectin. They were all weighed again on Days 7 and 14 and faecal sampled for a FECRT. The animals were killed on Days 14 or 15 and total worms counts undertaken on GI tracts and lungs. 2.3.1. FEC and FLC FEC was measured using a modified McMaster method (Stafford et al., 1994) to an accuracy of ±50 eggs per gram of faeces (epg). A modified Baermann’s technique (Thienpont et al., 1986) was used for FLC. 2.3.2. Gastro-intestinal worm counts The GI tract, minus the reticulo-rumen and omasum, was collected at slaughter, and the abomasum, small intestine (SI) and large intestine (LI) were tied off and stripped of all fat. The parasitological methods followed the World Association for the Advancement of Veterinary Parasitology (WAAVP) procedures for ruminants (Wood et al., 1995) except for enumeration of the early 4th stage larvae. Abomasa were washed out and two 2% aliquots of the contents saved and formalised. The washed abomasa were then individually incubated overnight in physiological saline (0.85% sodium chloride) at 37 ◦ C washed and sieved (38 micron aperture) and the sieved washings formalised. Small intestines were washed out and two 2% aliquots saved and formalised. Large intestines were washed out and two 10% aliquots saved and formalised. One aliquot each was counted in the case of the small and large intestines while both aliquots were counted in the case of the abomasa.

2.4. Pharmacokinetics Heparinised blood samples were cooled, spun down and plasma taken off and frozen within 2 h of collection. The frozen samples were sent on dry ice to the AgResearch Grasslands laboratory and kept frozen until assayed. 2.4.1. Moxidectin assay The moxidectin analysis was performed as previously described (Leathwick and Miller, 2013). Briefly, 100 ␮l of plasma was placed in an eppendorf tube and 400 ␮l of cold acetonitrile was added to precipitate the protein. The sample was shaken briefly and then centrifuged for 5 min at 12,000 × g. A 200 ␮l aliquot of the supernatant was transferred to an auto-sampler vial for subsequent analysis. A 5 ␮l aliquot was injected onto an Agilent SB-C8 column held at 25 ◦ C and the moxidectin eluted by reversed-phase gradient elution using 0.1% formic acid in MilliQ® water and 0.1% formic acid in acetonitrile (LiChroSolv grade, Merck, New Zealand). Ions were generated using positive electrospray ionisation on the mass spectrometer and detected with a Thermo TSQ Access Max triple quadrupole, monitoring the SRM parent/daughter ion transitions of 640.3 m/z to 498.3 m/z respectively. Full details of the detection limits, linearity and recoveries are reported elsewhere (Hughes et al., 2013). 2.4.2. Abamectin assay The abamectin analysis was performed using highpressure liquid chromatography (HPLC) with fluorescence detection with modifications of methods described previously (Alvinerie et al., 1995; Zele et al., 2011). Briefly, 500 ␮l of plasma was placed in an eppendorf tube and 500 ␮l of cold acetonitrile was added to precipitate the protein. The sample was shaken briefly and then centrifuged for 5 min at 12,000 × g. The supernatant was then transferred to a pre-conditioned C18 cartridge and washed with 1 ml of 50:50 acetonitrile:water and then briefly vacuum dried. The abamectin was then eluted with 1.5 ml of methanol into a 2 ml auto-sampler vial, dried under a constant stream of nitrogen at 60 ◦ C, derivitised, and a 10 ␮l aliquot resolved

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using reversed-phase gradient elution on a C18 column and detected using fluorescence detection (365 Ex, 475 Em). 2.5. Efficacy calculations Arithmetic efficacy (AE) % was calculated according to WAAVP guidelines (Wood et al., 1995). Control group mean-treatment group mean Control group mean%

Efficacy% =

FECRT: The FECR efficacy for each treatment group was calculated according to equations as follows: FECRa (McKenna, 1990):



100 × 1 −

 Mean epg post-treatment  Mean epg pre-treatment

animal was the only treated animal (1/36) to have any T. axei (n = 10), O. venulosum (n = 130) or lungworms (n = 640) at slaughter and it was excluded from analyses on the basis that the subcutaneous injection appears to have been administered incorrectly. This conclusion was also supported by finding negligible plasma concentrations of abamectin in samples from this animal (see below). Arithmetic efficacy: Moxidectin injection had the highest AE against adult Ostertagia-type nematodes (77.4%), and it was significantly more efficacious than both oral moxidectin (26.0%) and pour-on moxidectin (27.6%) (P < 0.01) (Table 1). Abamectin injection had an AE of 72.4% against adult Ostertagia-type nematodes, and was significantly more efficacious than abamectin pour-on (34.1%) (P < 0.05), but not oral abamectin (70.1%). There was no significant difference in efficacy between abamectin and moxidectin

FECRb (Presidente, 1985):



100 × 1 −

 Mean epg post-treatment   Mean epg pre-Control  Mean epg pre-treatment

Mean epg post-Control

FECRc (Vizard and Wallace, 1987)



100 × 1 −

 Mean epg post-treatment  Mean epg pre-Control

2.6. Statistical analysis Analysis of covariance of the seven treatment groups was performed, with the covariates being the source of animals, total Ostertagia-type, Ostertagia-type early 4th stage larvae (Ost EL4), lungworm, log (Ostertagia spp + 1) and FEC at Day 0, 7 and 14. Pharmacokinetic analyses included calculation of Area Under the Curve (AUC), estimates of total absorption of drug after oral and pour-on administration assuming 100% absorption from subcutaneous injection, and calculation of half-life of the drugs using a two-compartment model (Gibaldi and Perrier, 1982). 3. Results 3.1. Parasite numbers and species The most common parasites present in the control group animals were lungworm, D. eckerti (Johnson et al., 2001) and Ostertagia-type nematodes in the abomasum (Table 1). There were small numbers (20–100) of Oesophagostomum venulosum in the LI. Only one animal (see below) had a small number of Trichostrongylus axei and there were no other nematodes found. Treated animals had only Ostertagia-type nematodes. The Ostertagia-type species in the Control group included Spiculopteragia asymmetrica (56.4%) (including 1% Spiculopteragia quadrispiculata, which is regarded as a morphological variant), Ostertagia leptospicularis (31.8%) (including 1% Ostertagia kolchida), Spiculopteragia spiculoptera (11.8%) and Ostertagia ostertagi (2%). 3.2. Efficacy One deer (#433) in the abamectin injection group had unusual results, which are highlighted in Table 1. This

injections or pour-ons. There was a surprising difference in efficacy between oral abamectin (70.1%) and oral moxidectin (26.0%) (P < 0.01). Moxidectin and abamectin injections had similar efficacy (86.9 and 74.9%, respectively) against immature (EL-4) Ostertagia-type nematodes and these were significantly greater than moxidectin and abamectin oral (21.5 and 36%, respectively) and moxidectin pour-on (3.7%) treatments (P < 0.01), while abamectin pour-on had an efficacy of 48.5%. 3.3. Efficacy as indicated by FECRT There was considerable variability in FEC on Day 0, ranging from 0 to 100 epg with the majority having 0 epg (20 × 0, 10 × 50 and 4 × 100 epg) and the pre-treatment group means ranging from 10–50 epg. In comparison with the efficacy based on nematode counts at slaughter, the FECRT at Day 7 and 14 generally overestimated the efficacy of both anthelmintics for all three routes of administration, except for the McKenna formula that underestimated efficacy (0%) for four treatments when the pre and post treatment mean FEC were the same (Table 2). The FECR results indicated that the moxidectin and abamectin injections were 100% effective, the pour-ons were 0–100% effective and the oral treatments were 0–97% effective at Days 7 and 14. The faecal larval count reduction test (FLCRT) for lungworm was more reliable in that there was a good agreement between FLC and numbers of adult lungworm present in Control animals at slaughter, while the zero FLC of all treated animals on Day 14 reflected the zero lungworm count at slaughter (except for the excluded animal #433, which had FEC of 63, 51 and 29 for Days 0, 7 and 14 and had 640 Dictyocaulus at slaughter, which supports the conclusion that this animal had not received an appropriate dose rate of anthelmintic). There was a significant Pearson’s correlation (R = 0.84, P = 0.019) between the individual adult Ostertagia-type nematode count and the FEC in the untreated control deer

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Table 1 Individual and group mean counts and arithmetic efficacy (AE) % for Ostertagia-type adults (Ost adult), Late L4 (Ost LL4), Early L4 (Ost EL4), adult Trichostrongylus axei (T. axei), lungworm (LW) and Oesophagostomum venulosum (Oes) nematodes in the untreated Control group and the six treatment groups. Group

Tag

Ost adult

Control

301 348 419 428 439 Mean

1070 1330 2245 700 975 1264

Moxidectin injection

313 362 431 446 456 Mean AE%

Moxidectin oral

Ost EL4

T. axei

LW

Oes

0 0 0 0 0 0

118 236 120 0 99 114.6

0 0 0 0 0 0

2850 2750 5280 1550 2350 2956

20 100 30 60 40 50

130 245 490 175 390 286 77.4

25 0 0 0 0 5

0 39 0 0 36 15 86.9

0 0 0 0 0 0

0 0 0 0 0 0 100

0 0 0 0 0 0 100

302 306 365 437 461 Mean AE%

1400 825 375 730 1345 935 26.0

0 10 0 0 0 2

171 115 87 41 36 90 21.5

0 0 0 0 0 0

0 0 0 0 0 0 100

0 0 0 0 0 0 100

Moxidectin pour-on

310 436 438 457 480 Mean AE%

1575 610 640 670 1080 915 27.6

0 0 0 0 0 0

0 128 158 43 223 110.4 3.7

0 0 0 0 0 0

0 0 0 0 0 0 100

0 0 0 0 0 0 100

Abamectin injectiona

304 316 435 445 Mean AE%

325 970 25 75 349 72.4

0 0 0 0 0

0 115 0 0 28.8 74.9

0 0 0 0 0

0 0 0 0 0 100

0 0 0 0 0 100

Abamectin oral

338 417 455 458 474 Mean AE%

210 825 155 575 125 378 70.1

0 0 0 0 0 0

0 293 0 34 40 73.4 36.0

0 0 0 0 0 0

0 0 0 0 0 0 100

0 0 0 0 0 0 100

Abamectin pour-on

318 326 422 432 482 Mean AE%

575 725 995 700 1170 833 34.1

0 0 0 0 0 0

0 38 0 257 0 59 48.5

0 0 0 0 0 0

0 0 0 0 0 0 100

0 0 0 0 0 0 100

a

Ost LL4

Tag 344 excluded.

at slaughter (Day 14/15), but this relationship was not present in the 35 treated deer, with the majority of animals having a zero egg count despite having worm counts up to 1575, suggesting that the treatment had suppressed egg production without killing all the worms (Fig. 1A and B). 3.4. Efficacy against Ostertagia-type species The major and minor morphs (≤1%) were combined and the O. ostertagi (<2%) were not considered in this analysis. Although the results are not conclusive, there is a strong suggestion that S. asymmetrica was more resistant to

moxidectin than the other species because the percentage of this species in the residual nematodes after treatment was considerably higher in all three treated groups (MI 94.7%, MO 72.7%, MP 66.2%) compared with the untreated control (56.4%) (Table 3). This was most marked in the moxidectin injection group, which had the highest overall efficacy, although it also had the smallest number available for species differentiation (n = 20). The difference was not as marked in the abamectin treated groups (AI 60%, AO 79.4%, AP 51.9%). Overall the proportion of S. asymmetrica rose to 69.2% in the 6 treated groups combined compared with 56.4% in the Control group.

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Total adult Ostertagia-type

R2=0.84, P=0.019

2000 1500 1000 500 0 0

100

200 FEC

300

400

3000 Total adult Ostertagia-type

72.4 70.1 34.1 349 378 833 100% 78.2% 92.7% 100% 92.7% 92.7% 100% 0% 0% 0 30 10 100% 90% 90% 100% 97% 90% 100% 67% 0% 37.5 30 10 Abamectin injection Abamectin oral Abamectin pour-on

0 10 10

77.4 26.0 27.6 1264 286 935 915 100% 84.5% 100% 100% 92.7% 100% 100% 0% 100% 137.5 0 20 0 100% 90% 70% 100% 95% 94% 100% 50% 40% 100 0 10 30 10 30 20 50 Control Moxidectin injection Moxidectin oral Moxidectin pour-on

Control up p grou Controlgro

2500

Anthelmintic treated

2500 2000 1500 1000 500 0

Day 0 mean FEC

Day 7 mean FEC

Day 7 FECRa McK

Day 7 FECRb Pres

Day 7 FECRc Viz.

Day 14 mean FEC

Day 14 FECRa McK

Day 14 FECRb Pres

Day 14 FECRc Viz.

Mean Ost w/c

Efficacy (Ost w/c)

3000

Group

Table 2 Apparent efficacy of different anthelmintic treatments based on faecal egg count reduction tests (FECRT) at Day 0, 7 and 14 using three different formulas (FECRa, McKenna; FECRb, Presidente; FECRc, Vizard) compared with efficacy based on worm counts (w/c) at slaughter on Day 14/15.

86

0

100

200 FEC

300

400

Fig. 1. Relationship between abomasal Ostertagia-type nematode counts and FEC at slaughter in the six untreated Control deer (A) compared with 35 anthelmintic treated deer (B).

3.5. Pharmacokinetics Peak plasma concentrations (Cmax ) of both moxidectin and abamectin injections and oral treatments were highest at the time of the first post-treatment sample (12 h) and both pour-on treatments had peak plasma levels 5 days post treatment. Cmax mean plasma levels (ng/ml) for moxidectin were 71.8 (MI), 8.3 (MO) and 0.4 (MP) and for abamectin were 62.1 (AI), 30.3 (AO) and 10.0 (AP) (Table 4 and Fig. 2). Moxidectin and abamectin levels in plasma from the untreated control deer were minimal, with all readings being within the margin of error for the quantification of drug concentration in the assay. Animal #433 in the abamectin injection group had abamectin plasma levels in the same range as the Control animals. AUC estimates were 106.6, 12.9, 6.1 and 0.5 for the MI, MO, MP and Control groups, respectively, and 162.7, 57.5, 74.3 and 4.8 for the AI, AO, AP and Control groups, respectively. Assuming that 100% was absorbed from the subcutaneous injection, these estimates imply that on average for moxidectin approximately 13.9% was absorbed from the oral dose and 5.7% from the pour-on dose. For abamectin approximately 33.4% and 51% were absorbed from oral and pour-on doses of abamectin, respectively.

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Table 3 The number (and percentage) of each male Ostertagia-type species in a representative sample of nematodes collected from the abomasal washings and incubated abomasal washings of the untreated Control group and the six treatment groups, and the overall total (and percentage) for the treated groups for each species. Spiculopteragia asymmetricaa

Ostertagia lleptospicularisb

Spiculopteragia spiculoptera

No.

Total

%

No.

%

No.

%

Control Moxidectin injection Moxidectin oral Moxidectin pour-on

62 18 80 43

56.4 94.7 72.7 66.2

35 1 19 18

31.8 5.3 17.3 27.7

13 0 11 4

11.8 0.0 10.0 6.2

110 19 110 65

Abamectin injection Abamectin oral Abamectin pour-on

9 27 27

60.0 79.4 51.9

5 5 20

33.3 14.7 38.5

1 2 5

6.7 5.9 9.6

15 34 52

204

69.2

68

23.1

23

7.8

295

Total treated a b

Including 1% S. quadrispiculata. Including 1% O. kolchida.

Table 4 Mean estimates of maximum plasma concentration (Cmax ) in ng/ml, time of Cmax (Tmax ) and area under the curve (AUC) of moxidectin and abamectin for groups of deer treated by three different routes of administration and untreated control deer. Treatment

Cmax

Tmax

AUC

AUC min

AUC max

Moxidectin injection Moxidectin oral Moxidectin pour-on Control

71.8 8.3 0.4 0.04

0.5 days 0.5 days 5 days 0.5 days

106.6 12.9 6.1 0.5

83.3 7.7 3.4 0.2

125.6 18.5 9.2 0.8

Abamectin injection Abamectin oral Abamectin pour-on Control

62.1 30.3 10.0 0.5

0.5 days 0.5 days 5 days 7 days

162.7 57.5 74.3 4.8

66.0 46.9 3.4 3.5

296.7 76.7 119.3 6.1

These estimates suggest that the pharmacokinetics of moxidectin and abamectin are quite different. For estimating the half-life of moxidectin after subcutaneous injection, a two compartment model was fitted and the half- life was estimated to be 0.51 days, but the predicted rate of decline was swifter than the actual data showed. However, the abamectin half-life after subcutaneous injection was estimated to be 4.6 days and much more closely fitted a two compartment model. There was a significant inverse relationship between the number of adult abomasal nematodes at slaughter and the individual peak plasma levels of the 15 deer treated with moxidectin (P < 0.001) and the 14 treated with abamectin (P = 0.015), and fitted curved regression lines (R2 = 0.656 and R2 = 0.335, respectively) after log-log transformation of the data (Fig. 3). 4. Discussion This study was carried out in the autumn when peak levels of larval challenge are present and most young newly weaned deer are still susceptible to parasites. This is also the period when anthelmintics are most commonly used on deer farms in New Zealand. The study animals had moderate to high burdens of lungworm and abomasal nematodes, which are the most important nematodes infecting deer. It appears that D. eckerti and O. venulosum are still very susceptible to ML anthelmintics after 30 years of use of ivermectin and moxidectin. However, there was clear evidence of serious ML resistance in Ostertagia-type parasites. S. asymmetrica (including minor morph S. quadrispiculata)

Variance 243.8 16.6 4.4 0.1 7313 134 1983 1

SD 15.6 4.1 2.1 0.2 85.5 11.6 44.5 1.1

was the most common species in the Control animals at slaughter and there is a suggestion that it is the most resistant species on this farm since the proportions were higher after treatment in all three moxidectin treated groups. However this increase in the proportion of S. asymmetrica was not as marked after treatment with abamectin. Of the three routes of administration, injection gave the highest efficacy for both moxidectin and abamectin, and the arithmetic efficacies were very similar. Pour-on treatments of moxidectin and abamectin also had very similar efficacies, but were significantly lower than equivalent injectible treatments. There was a surprising difference in efficacy between the two oral treatments, with oral abamectin significantly higher than oral moxidectin. According to the manufacturers’ labels the formulations were very similar, except that the oral moxidectin used was plain while the only abamectin product available at the time was “Hi Mineral”, which had added minerals; each 10 ml dose contained 10 mg iodine, 5 mg selenium, 2 mg cobalt, 21 mg copper and 6 mg zinc. It has been shown that 2% copper sulphate and 2% cobalt sulphate induced oesophageal groove closure in the majority of young sheep and similar copper sulphate solutions caused oesophageal groove closure in 70% of cattle (Carruthers et al., 1994). This reflex, which directs liquid in the oesophagus directly into the abomasum, rather than the rumen, is elicited by receptors in the mouth and pharynx, is well developed in suckling ruminant neonates but becomes less reliable in older animals. It has been reported that when oxfendazole was administered directly into the abomasum in sheep it resulted in the

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90

Moxidectin ng/ml plasma

80 70 Moxidectin

60

Injection

50

Oral

40

Pour-on

30

Control

20 10 0 0

24

48

72

96

120

144 168 192 216 Hours after treatment

240

264

288

312

336

80

Abamectin ng/ml plasma

70 Abamectin

60

Injection

50

Oral

40

Pour-on

30

Control

20 10 0 0

24

48

72

96

120

144 168 192 216 Hours after treatment

240

264

288

312

336

Fig. 2. Mean plasma moxidectin and abamectin (±SE) levels (ng/ml) over 14 days for deer treated by three different routes of administration and untreated control deer.

peak plasma oxfendazole concentration occurring sooner and the AUC of plasma oxfendazole was reduced when compared with intra-ruminal administration (Prichard and Hennessy, 1981). Furthermore, there is evidence that it is beneficial for benzimidazoles to be deposited in the rumen to increase total absorption of drug and increase efficacy (Hennessy, 1993). However, the opposite may apply for ML drugs. Deposition in the rumen appears to slow the absorption of ivermectin and doramectin, while rumen bypass appears to result in an earlier Tmax for orally administered moxidectin (Hennessy and Alvinerie, 2002). Similarly, it has been reported that there was a 3–4 fold increase in Cmax and AUC for ivermectin after intra-abomasal administration compared with intra-ruminal administration and Tmax was reduced from 23 to 4 h (Prichard, 1985). In the present study, the first blood samples were taken 12 h after treatment and the highest plasma levels for both oral anthelmintics were measured at this 12 h sampling, at which time the Tmax for oral abamectin (30.3 ng/ml) was significantly higher than for oral moxidectin (8.3 ng/ml). A higher Tmax is likely to be more effective against partially resistant parasites, which may explain the higher efficacy of the Hi Min abamectin over the plain

moxidectin oral drench. The AUC for oral abamectin was also over 4 times greater than for oral moxidectin. Further research is planned to investigate the possible involvement of rumen bypass on the efficacy of orally administered ML in deer. The relative efficacy of moxidectin oral versus injection may depend on the species of animals and the predominant species of nematode. A study in sheep, found that moxidectin injection was more effective against Trichostrongylus colubriformis than moxidectin oral, although both were very effective against Teladorsagia circumcincta and Haemonchus contortus (Kerboeuf et al., 1994). A recent study in cattle (Leathwick and Miller, 2013) found that the reduction in FEC was significantly greater after treatment with moxidectin oral (91.1%) than moxidectin injection (55.5%) and moxidectin pour-on (51.3%). Low efficacies were invariably against Cooperia oncophora. The oral treatments were significantly less variable in efficacy than the injection and pour-on treatments. In general, ML administered by injection reach a higher Cmax (Herd et al., 1996; Vercruysse and Rew, 2002) and have higher efficacy than when the same active is administered as a pour-on (Sargison et al., 2009; Whang et al., 1994).

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Adult abomasal nematodes

1600

89

Moxidectin

1400 1200 1000 800 600

y = 923.99e-0.018x R² = 0.6563

400 200 0 0

20

Adult abomasal nematodes

1600

40 60 80 Moxidectin ug/ml plasma

100

120

Abamectin

1400 1200 1000 y = 4439.3x-0.774 R² = 0.355

800 600 400 200 0 0

20

40 60 80 Abamectin ug/ml plasma

100

120

Fig. 3. Relationship between the numbers of adult abomasal nematodes remaining at slaughter and the peak plasma levels of moxidectin and abamectin with the three treatment methods for each combined, with fitted curved lines of log-log regression and R2 values (P < 0.001 and P = 0.015, respectively).

There has been concern raised about the inconsistent efficacy seen with pour-on ML anthelmintics in cattle and this has been attributed to the poor absorption and lower Cmax in plasma compared with oral or injection counterparts (Vercruysse and Rew, 2002), as well as poor efficacy against dose-limiting nematode parasites such as Cooperia and Nematodirus spp. (Sargison, 2011). In the present study these latter species were not present, while Ostertagiatype nematodes, which are the most important GI species in farmed deer, were showing the greatest resistance and moxidectin injection was significantly more efficacious than moxidectin pour-on and oral moxidectin against these parasites. Clearly there has been a dramatic decline in efficacy of pour-on moxidectin on the study farm since the previous efficacy trial in 1996 (Mackintosh et al., 1997), which had similar findings to another study in 1994 (Middelberg, 1994). In New Zealand and internationally, anthelmintic resistance has been a growing problem in the sheep and cattle industries for a number of years (Leathwick et al., 2001; Sutherland and Leathwick, 2010), but it has only relatively recently been detected in the deer industry. A study 10 years ago found a mean efficacy of 94% for moxidectin pour-on, 68% for pour-on ivermectin and 31% for

oral ivermectin against adult Ostertagia-type nematodes on a North Island deer farm and the authors concluded that this was evidence of anthelmintic resistance developing (Hoskin et al., 2005). Subsequently, other studies have demonstrated anthelmintic resistance in deer on two farms in Southland, with moxidectin pour-on having poorer efficacy (71.2% and 19.3%) than moxidectin injection (83.5% and 87.1%, respectively) (Lawrence, 2011). In the current study, efficacy against lungworm remained at 100%, which may be due to the susceptibility of the parasite and the high degree of vascularisation in the lungs. It has been suggested there is some enterohepatic recycling of ML and they are largely excreted via the GI tract resulting in high concentrations of ML in intestinal mucous after treatment (Vercruysse and Rew, 2002), which may explain the high efficacy against intestinal nematodes, including Oesophagostomum spp. Various methods of calculating anthelmintic efficacy in the FECRT have been discussed previously (Miller et al., 2006), but irrespective of the method, the FECRT conducted in the present study was very inconsistent at Days 7 and 14 and tended to overestimate the efficacy of treatments compared with the efficacy derived from nematode counts at slaughter. On only one occasion (abamectin oral) the Vizard

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estimate of FECR was similar to the worm count efficacy. The discrepancies for the FECR were greater at Day 7 than at Day 14 and the overestimates were greatest for pouron treatments. These results suggest that the sublethal treatments caused temporary suppression of egg production in the nematodes that survived, as reported previously (Mason and McKay, 2006). The increase in the mean FEC of the Control group of 8 epg on Day 0 to 137.5 epg on Day 14 probably also contributed to some anomalous FECR calculations, especially using the Presidente formula. This rise was probably due to the maturation of nematodes that developed over this period from the dose of infected larvae administered on day −21 and any infective larvae picked up from pasture in the weeks prior to the study starting. It has been suggested that the inaccuracy of the FECRT is due to a poor correlation between the FEC and the actual number of GI nematodes present (Taylor et al., 2002). The effect may have been further increased by the presence of Oesophagostomum spp., which tend to be very fecund and would have been contributing to the FEC prior to treatment in all groups, but not in the treated groups on Days 7 and 14. However in this study we demonstrated a good correlation between FEC and Ostertagia-type nematode count in the untreated animals at slaughter compared with a poor correlation in treated animals, which supports the conclusion that treatment suppressed egg production in surviving nematodes. Other studies have also recorded significantly lower FEC for samples collected 7 days after treatment compared with FEC on day 14 and 21 (Leathwick and Miller, 2013). This effect would have been exacerbated by the high degree of anthelmintic resistance on this farm. Similarly, a previous study showed that the FECRT overestimated the efficacy of ivermectin and moxidectin treatment in red deer (Hoskin et al., 2005). These authors stated that “this study suggests that nematode recovery from organs following necropsy, rather than a FECRT, should be used to determine efficacy of anthelmintics in farmed deer, particularly if pour-on anthelmintics are used”. Clearly the different routes of administration resulted in big differences in plasma concentrations, Cmax , Tmax and AUC, which then may have influenced their efficacy. As in cattle (Sallovitz et al., 2002), moxidectin and abamectin pour-ons administered at 0.5 mg/kg resulted in significantly lower plasma concentrations than the subcutaneous injection of each anthelmintic at 0.2 mg/kg, while oral moxidectin and abamectin were intermediate. The Cmax at 12 h for moxidectin and abamectin injection were very similar, but after this the moxidectin plasma concentrations declined quickly and had reached 1.2 ng/ml by day 7, whereas abamectin took 14 days to decline to a similar level and had an AUC that was 77% higher. Nevertheless the efficacy of both injections was very similar. Oral abamectin had a Cmax over 3 times and an AUC over 4 times that of oral moxidectin, which might partially explain why the oral abamectin efficacy was significantly higher than oral moxidectin against Ostertagia-type nematodes. The Cmax and AUC of abamectin pour-on were over 12 times higher than those of moxidectin pour-on, but the efficacy was similarly very poor for both, indicating that the plasma concentrations were insufficient for effective nematocidal activity for both. It appears from these results that

Cmax may be more important than AUC, because the AUC for abamectin pour-on was greater than abamectin oral. This data also suggests that the minimum lethal plasma concentration for these resistant Ostertagia-type nematodes is now substantially greater than the 9–10 ng/ml achieved by moxidectin oral and abamectin pour-on. Studies 15–20 years ago in cattle suggested that moxidectin plasma concentrations of 0.5–1.0 ng/ml were sufficient for optimal nematocidal activity (Lifschitz et al., 1999) and were probably similar for deer at that time. In the present study moxidectin pour-on achieved these concentrations 5–7 days after treatment but the treatment was ineffective. The formulation of Cydectin Pour-on has not changed in the last 20 years and treatment of farmed red deer 20 years ago would have resulted in very similar moxidectin plasma concentrations to those found in this study, which were highly effective, suggesting that deer parasites have subsequently developed resistance, and now require much higher plasma concentrations. It is likely that the prolonged low plasma concentrations resulting from pour-on application have accelerated this development of resistance. Even concentrations of 68–71 ng/ml achieved by abamectin and moxidectin injections were <80% effective, which is far from satisfactory. The significant inverse relationship between the number of adult abomasal nematodes at slaughter and the individual peak plasma levels for both actives (Fig. 3) demonstrate that higher peak ML concentration are more effective at eliminating Ostertagia-type nematodes. Moxidectin has high lipophilicity (Sallovitz et al., 2003) and the rapid decline in plasma levels after injection and the much lower peaks after oral and pour-on treatment are likely to have been due to the rapid partitioning of the drug from plasma into fat reserves. It may also explain the reason why use of a two-compartment model overestimated the half-life of moxidectin, because the actual plasma concentration declined more quickly than predicted, suggesting that body fat/lipid acted as a third compartment and absorbed the circulating moxidectin quickly. However, this may also slow the rate of elimination because the drug must be absorbed back into blood circulation before it is excreted. Abamectin has lower lipid solubility than many of the other ML (Cerkvenik-Flajs et al., 2007) and the plasma concentrations more closely fitted a two compartment model and the half life after subcutaneous injection was estimated to be 4.6 days, which was close to the decline of actual plasma concentrations with time. This suggests that more abamectin remained in circulation, rather than being absorbed by lipid/fat reserves, and the decline in plasma concentration was due to excretion by first order kinetics; i.e. the decline in the mean log plasma levels was a straight line. A pharmacokinetic study of abamectin in fallow deer (Dama dama), which are not closely related to red deer, observed a Cmax of 120 ng/ml 24 h after subcutaneous administration of 0.2 mg/kg, which was almost twice the Cmax found in this study (62.1 ng/ml), and the authors observed that it is almost four times higher than in sheep (30.9 ng/ml) (Cerkvenik-Flajs et al., 2007), two times higher than in cattle (54.1 ng/ml) (Borges et al., 2007) and four times higher than in beef calves (25.4 ng/ml) (Lifschitz

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et al., 2004). They also found that the plasma concentrations over time were most adequately described with a two-compartment model (Zele et al., 2011). The results of this study demonstrate that significant anthelmintic resistance to moxidectin and abamectin has developed over the last 20 years on this deer farm, especially to pour-ons. The injection was the most effective route of administration in young deer for both anthelmintics, although <80% efficacious. The FECRT tended to overestimate the efficacy of the treatments compared with nematode counts at slaughter. The addition of minerals to oral ML anthelmintics needs to be investigated further. Conflict of interest statement There are no potential conflicts of interest associated with this study. The manufacturers supplied the anthelmintics used in the study, but they had no part in funding, planning, analysing or reporting the results of the study. Acknowledgements We are grateful to the staff at AgResearch Invermay who assisted with the field work, and at Gribbles Invermay who conducted the FEC and FLC. This project was funded by DEEResearch and AgResearch Core Funding Contract A19114. Product was supplied by Zoetis and Merial NZ Ltd. References Alvinerie, M., Sutra, J.F., Badri, M., Galtier, P., 1995. Determination of moxidectin in plasma by high-performance liquid chromatography with automated solid-phase extraction and fluorescence detection. Journal of Chromatography B: Biomedical Sciences and Applications 674, 119–124. Borges, F.A., Cho, H.S., Santos, E., Oliveira, G.P., Costa, A.J., 2007. Pharmacokinetics of a new long acting endectocide formulation containing 2.25% ivermectin and 1.25% abamectin in cattle. Journal of Veterinary Pharmacology and Therapeutics 30, 62–67. Carruthers, V.R., Phipps, D.E., Bakker, R.J., 1994. The effect on oesophageal groove closure of water and mineral solutions drenched to cows. Proceedings of the New Zealand Society of Animal Production 54, 23–25. Castillo-Alcala, F., Wilson, P.R., Pomroy, W.E., Hoskin, S.O., 2007. A survey of anthelmintic use and internal parasite control in farmed deer in New Zealand. New Zealand Veterinary Journal 55, 87–93. Cerkvenik-Flajs, V., Grabnar, I., Koˇzuh Erˇzen, N., Marc, I., Antonic´ı, J., Vergles-Rataj, A., Kuˇzner, J., Pogacnik, M., 2007. Kinetics of abamectin disposition in blood plasma and milk of lactating dairy sheep and suckling lambs. Journal of Agricultural and Food Chemistry 55, 9733–9738. Gibaldi, M., Perrier, D., 1982. Pharmacokinetics, Second Edition (Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs). Informa Healthcare, USA. Hennessy, D.R., 1993. Pharmacokinetic disposition of benzimidazole drugs in the ruminant gastrointestinal tract. Parasitology Today 9, 329–333. Hennessy, D.R., Alvinerie, M.R., 2002. Pharmacokinetics of the macrocycic lactones: conventional wisdom and new paradigms. In: Vercruysse, J.A.R.R.S. (Ed.), Macrocyclic Lactones in Antiparasitic Therapy. CAB International, Wallingford, UK, pp. 97–123. Herd, R.P., Sams, R.A., Ashcraft, S.M., 1996. Persistence of ivermectin in plasma and faeces following treatment of cows with ivermectin sustained-release, pour-on or injectable formulations. International Journal for Parasitology 26, 1087–1093. Hoskin, S.O., Pomroy, W.E., Wilson, P.R., Ondris, M., Mason, P., 2005. The efficacy of oral ivermectin, pour-on ivermectin and pour-on

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Vercruysse, J., Rew, R.S., 2002. General efficacy of the macrocyclic lactones to control parasites of cattle. In: Vercruysse, J., Rew, R.S. (Eds.), Macrocyclic Lactones in Antiparasitic Therapy. CABI Publishing, Oxford, pp. 185–222. Vizard, A.L., Wallace, R.J., 1987. A simplified faecal egg count reduction test. Australian Veterinary Journal 64, 109–111. Waldrup, K.A., Mackintosh, C.G., Duffy, M.S., Labes, R.E., Johnstone, P.D., Taylor, M.J., Murphy, A.W., 1998. The efficacy of a pour-on formulation of moxidectin in young red and wapiti-hybrid deer. New Zealand Veterinary Journal 46, 182–185. Whang, E.M., Bauer, C., Kollmann, D., Bürger, H.J., 1994. Efficacy of two formulations (‘injectable’ and ‘pour on’) of moxidectin against gastrointestinal nematode infections in grazing cattle. Veterinary Parasitology 51, 271–281. Wood, I.B., Amaral, N.K., Bairden, K., Duncan, J.L., Kassai, T., Malone, J.B.J., Pankavich, J.A., Reinecke, R.K., Slocombe, O., Taylor, S.M., Vercruysse, J., 1995. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) second edition of guidelines for evaluating the efficacy of anthelmintics in ruminants (bovine, ovine, caprine). Veterinary Parasitology 58, 181–213. Zele, D., Tavcar-Kalcher, G., Kobal, S., Vengust, G., Vengust, A., Grabnar, I., 2011. Plasma pharmacokinetics of abamectin in fallow deer (Cervus dama dama) following subcutaneous administration. Journal of Veterinary Pharmacology and Therapeutics 34, 455–459.