Oliver Am

Drenching adult ewes: Implications of anthelmintic treatments pre- and post-lambing on the development of anthelmintic resistance

Abstract AIMS: To test the hypothesis that peri-parturient anthelmintic treatment of adult ewes, either pre-lambing with a control- led-release capsule (CRC) or at tail-docking with a short-acting oral formulation, would increase the rate of development of anthelmintic resistance, as compared to not drenching ewes and giving an additional drench to lambs in the autumn. Also, to evaluate the potential of routinely leaving 15% of the heaviest lambs untreated when drenching, as a means of slowing the development of anthelmintic resistance. METHODS: A replicated farmlet trial was run from 1999– 2004. Eleven farmlets, each consisting of five paddocks, were initially seeded with Ostertagia (Teladorsagia) circumcincta and Trichostrongylus colubriformis parasites, these being a mixture of albendazole-susceptible and -resistant isolates to yield a 96% reduction in faecal nematode egg count (FEC) on drenching. Four prescriptive drenching regimes were applied; Treatments 1–3 were replicated three times and Treatment 4 twice. Treatments were as follows. Treatment 1: Ewes were given an albendazole CRC pre-lambing, and any ewes exceeding 65 kg liveweight were given two capsules simultaneously; lambs were given a five-drench preventive programme of treatments, orally, of albendazole on Days 0, 21, 42, 70 and 98 after weaning. Treatment 2: Ewes were given a single oral treatment of albenda- zole at docking (2–3 weeks after lambing), and lambs were given the same five-drench preventive programme as in Treatment 1. Treatment 3: Ewes remained untreated, while lambs were given a six-drench preventive programme of treatments, orally, of al- bendazole on Days 0, 21, 42, 70, 98 and 126 after weaning. Treatment 4: Ewes remained untreated, while lambs were given the same six-drench preventive programme as in Treatment 3, but the heaviest 15% of lambs were left untreated each time. Albendazole-resistance status was measured at least twice-yearly, using faecal egg count reduction tests (FECRTs) and larval development assays (LDA). In addition, controlled slaughter of drenched and undrenched tracer lambs was undertaken in the last 3 years. RESULTS: Resistance to albendazole increased most rapidly in Treatment 1, as measured by FECRT and LDA results, and worm burdens in tracer lambs. In Treatment 2, resistance developed slower than in Treatment 1 but faster than in Treat- ments 3 and 4, as measured by LDA; resistance in Treatment 2 developed more quickly than in Treatment 4, as measured by FECRTs. There was no significant difference between Treatments 3 and 4, although this approached significance in Ostertagia spp, as measured by LDA. CONCLUSIONS: Anthelmintic treatments to adult ewes around lambing time are likely to be more selective for resistance than additional treatments administered to lambs in the autumn. Farmers wishing to slow the emergence of anthelmintic resistance on their farms should look to minimise the administration of peri-parturient treatment of ewes. A trend to slower development of resistance where a proportion of lambs were left untreated at each drench suggests further work on this aspect of management of resistance is warranted.

Selective and on-demand drenching of lambs: Impact on parasite populations and performance of lambs

Abstract AIM: To determine whether drenching regimes for lambs by which a proportion (10%) of the heaviest animals was selectively left untreated, or animals are only drenched ‘on demand’ when faecal nematode egg counts (FEC) exceeded a threshold level, would result in measurable increases in parasite larval challenge in the autumn and/or decreases in the performance of lambs. METHODS: A replicated study compared three drenching strategies in which mobs of lambs (n=360 in total) received either: a five-drench preventive programme, administered to all animals (Treatment 1); a five-drench preventive programme, but the 10% heaviest animals left untreated each time (Treatment 2); or drench treatments administered only when FEC exceeded 500 eggs per gram of faeces (epg) (Treatment 3). After the five-drench programme, animals in Treatments 1 and 2 were treated according to FEC as for Treatment 3. A triple-combination drench containing ivermectin, oxfendazole and levamisole, administered orally, was used for all treatments. There were nine farmlets, allowing three replicates of each treatment, in a completely randomised design. Parasite infestations on pasture were measured in autumn by pasture plucks, and worm burdens were monitored in tracer lambs, while the performance of lambs was assessed by liveweight gains, fleece weights, and body condition and dag scores. RESULTS: Increased numbers of Haemonchus contortus and Trichostrongylus colubriformis larvae on pasture were found in the autumn on farmlets treating selectively or on-demand (Treatments 2 and 3). No differences were detected in other parasite species. Mean liveweight gains did not differ between treatments but some differences were detected between drenched and undrenched lambs in Treatment 2. Mean body condition and mean dag scores of lambs in Treatment 3 tended to be lower and higher, respectively, than those of lambs in Treatment 1; Treatment 2 was generally intermediate. CONCLUSIONS: Drenching strategies for lambs designed to slow the development of anthelmintic resistance, by increasing the pool of susceptible worms available to dilute resistant survivors after treatment, resulted in increased numbers of H. contortus and T. colubriformis but not other species of parasite on pasture. The increased parasite challenge to lambs in the autumn was associated with small production losses, which may be acceptable to farmers wishing to implement such strategies. It is clear that further work is required on the interaction between management practices and the population dynamics of parasites, especially with regard to creating pools of susceptible genotypes to slow the development of drench resistance.

Managing anthelmintic resistance: Untreated adult ewes as a source of unselected parasites, and their role in reducing parasite populations

Abstract AIMS: To test the hypotheses that when untreated adult ewes are rotationally grazed (follow behind) on pastures after lambs receiving routine anthelmintic treatments, the ewes can function as a source of unselected parasites in refugia, capable of slowing the development of anthelmintic resistance, and suppress the build-up of parasites resulting from the development of anthelmintic resistance. METHODS: Firstly, the potential of untreated adult ewes to slow the development of anthelmintic resistance, and to suppress parasite populations under differing levels of anthelmintic efficacy, was investigated using a simulation model. Secondly, a field trial with three replicates of each treatment compared two grazing systems (lambs only vs lambs followed by ewes) and two types of anthelmintic, viz albendazole (ALB), to which resistance was present (faecal nematode egg count reduction (FECR)=57–59%) and ivermectin plus levamisole (IL), to which resistance was absent (FECR=97–99%), in a factorial treatment structure. Parasite populations were monitored using faecal nematode egg counts (FEC), faecal larval cultures, pasture larval sampling, and slaughter of tracer lambs. Animal performance was measured using liveweight, dag score, body condition score, and fleece weights. RESULTS: Model simulations indicated that parasites cycling in the untreated ewes could slow the development of resistance being selected for by the anthelmintic treatments given to lambs and this could occur without a nett increase in larval numbers on pasture. Further, as worm control in the lambs declined with increasing levels of anthelmintic resistance the ewes increasingly functioned as nett removers of parasite larvae, effectively reducing parasite population size. In the field trial, untreated adult ewes contributed to pasture infestations of most parasite species, but not Nematodirus spp. Parasite species on pasture and infecting lambs changed when ewes were present, but larval populations on pasture in the autumn were no greater than when lambs grazed alone. In the presence of anthelmintic resistance, parasite populations were reduced when ewes grazed in rotation with lambs, implicating the ewes as nett removers of parasite challenge. CONCLUSIONS: Untreated adult ewes were a source of unselected genotypes, capable of slowing the development of anthelmintic resistance in most, but not all, parasite species. Further, the potential of adult ewes to remove from pasture more parasite larvae than they contribute through faecal contamination indicates a potentially useful role in suppressing parasite populations, particularly when worm control in lambs is less effective as a result of anthelmintic resistance.

Drench-and-shift is a high-risk practice in the absence of refugia

Abstract AIM: To examine the effect of an anthelmintic treatment to lambs, followed immediately by a shift onto pastures with differing levels of larval contamination, on the development of anthelmintic resistance, in order to support recommendations to farmers regarding drench-and-shift practices for sustainable worm control. METHODS: Newly weaned Romney lambs (n=72) were dosed with third-stage infective larvae (L3) of two nematode parasite species, Teladorsagia (=Ostertagia) circumcincta and Trichostrongylus colubriformis, comprising benzimidazole-resistant and -susceptible isolates, calculated to yield, after treatment with albendazole, a 95% reduction in faecal nematode egg count (FEC). Once infections became patent (Day 0), lambs were randomised into nine groups of eight animals, treated with albendazole at the manufacturers recommended dose rate, and moved to individual pastures each previously prepared to have one of three different levels of parasite larval infestation (Treatment 1 = low contamination, Treatment 2 = medium contamination, and Treatment 3 = high contamination), and grazed on those pastures before receiving a second treatment with albendazole at Day 47. Anthelmintic resistance status in each group of lambs was measured using FEC reduction (FECR) and egghatch assays (EHA) after the first anthelmintic treatment, and FECR after the second treatment. RESULTS: Egg-hatch assays demonstrated significant differences between treatments. The concentration of anthelmintic required to kill 50% of the eggs (LC50) for Treatment 1, comprising the least contaminated pastures, was significantly higher than for Treatments 2 and 3 on Days 33 and 40. Treatment 1 also had a significantly lower FECR at the final anthelmintic treatment, and significantly lower FEC than the other two treatments from Days 26 to 47. CONCLUSIONS: The results demonstrated that the populations of T. circumcincta and T. colubriformis in lambs treated with anthelmintic had significantly higher levels of albendazole resistance at the end of the grazing period in lambs moved onto pastures with relatively low levels of parasite contamination than those moved onto pastures with relatively higher contamination, confirming drench-and-shift onto ‘clean’ pasture as a high-risk practice for the selection for anthelmintic resistance. While this does not necessarily preclude the use of this practice it does emphasise the importance of taking appropriate remedial action to minimise the risk.

Dynamics of the free-living stages of sheep intestinal parasites on pasture in the North Island of New Zealand. 2. Weather variables associated with development.

Abstract AIM: To identify weather variables associated with the development of eggs of Teladorsagia (=Ostertagia) circumcincta, Trichostrongylus colubriformis and Haemonchus contortus to third-stage infective larvae (L3) under a range of climatic conditions on pasture in the North Island of New Zealand. METHODS: Sheep faeces containing known numbers of eggs of all three nematode species were deposited on, or buried in, pasture plots at three sites, viz coastal Manawatu, Upper Hutt Valley, and East Cape hill country. Development was measured by recovering L3 from faeces, herbage and soil 28–31 days after deposition, on 13–18 occasions between January 2005 until July 2006. Weather data were recorded at each site, and the association between weather variables and number of L3 recovered was analysed using subsets regression to select best-fitting models from several candidate variables, after adjustment for efficiency of recovery of L3. A multiple linear regression model was then developed for each species, to select weather variables that had both significant and substantive effects on the number of L3 recovered. RESULTS: For all species, mean daily temperature was the bestpredictor of the number of L3 recovered (p=0.001). For T.circumcincta the final model included mean daily temperature and soil temperature (R2=51%), and for T. colubriformis the model only included mean daily temperature (R2=55%). For development of H. contortus, mean daily temperature was the most significant variable, but moisture in the form of rainfall entropy, i.e. the temporal distribution of rainfall, overthe first 14 days was also significant in the final model (R2=34%). CONCLUSIONS: Temperature was the most important determinant of developmental success of free-living nematodes on pasture at the study sites, and probably also for other parts ofNew Zealand with similar climates. Moisture was not significant in the development of T. circumcincta or T.colubriformis, implying that under the generally moist temperate climate in New Zealand, moisture is seldom limiting for development of these species. Haemonchus contortusappeared to be sensitive to moisture availability and precipitation in the first 14 days after deposition of faeces. The results of this study will be further developed as part of species-specific climate-driven models of parasite development in New Zealand.

Dynamics of the free-living stages of sheep intestinal parasites on pasture in the North Island of New Zealand. 1. Patterns of seasonal development

Abstract AIM: To describe the seasonal pattern of development of third-stage infective larvae (L3) from eggs of Teladorsagia (=Ostertagia) circumcincta, Trichostrongylus colubriformis and Haemonchus contortus on pasture in the North Island of New Zealand. METHODS: Sheep faeces containing known numbers of eggs of all three nematode species were deposited on, or buried in, pasture plots at three sites, viz coastal Manawatu, Upper Hutt Valley, and East Cape hill country. Development was measured by recovering L3 from faeces, herbage and soil 28–31 days after deposition on 13–18 occasions, between January 2005 and July 2006. Analysis of the number of larvae recovered used a mixed model including number of eggs deposited, weight of faeces recovered (an assumed indicator of earthworm activity), site, contamination date, and position of deposited faeces, i.e. on the surface or buried. RESULTS: There was a significant effect of contamination date on development of all three species, with maximum numbers ofL3 developing between late spring (November) and early autumn (March), and minimum numbers in June and July. There were large differences between species, with H. contortus exhibiting a long period (April to October) where development was close to zero, whereas T. circumcincta developed to some extent all year round. Development of T. colubriformis was intermediate between the other two species. Burying faeces containing nematode eggs increased the number of L3 recovered compared with surface deposition (p≤0.001), although there were a small number of exceptions involving only T. colubriformis. The weight of faeces recovered at harvest, which was assumed to be an indication of earthworm activity, was correlated with the number of L3 recovered for all species (p<0.001). In a separate analysis, earthworms were assumed tohave been active if <5 g faeces remained at harvest. Where this occurred, the number of L3 of T. colubriformis and T.circumcincta recovered was reduced by 56% and 58%, respectively (p<0.001). CONCLUSIONS: A marked seasonal pattern of development was observed for all three species, with the most larvae developing in spring-early autumn and the least in winter. This seasonal pattern was most pronounced in H. contortus and least obvious in T. circumcincta. Burying faeces containing eggs generally resulted in more L3 being recovered, whilst the apparent activity of earthworms resulted in fewer larvae being recovered.

A survey of the prevalence of Nematodirus spathiger and N. filicollis on farms in the North and South Islands of New Zealand

Abstract AIM: To compare the prevalence of Nematodirus spathiger and N. filicollis on a sample of farms in the lower South Island with the lower and central North Island of New Zealand by way of a presence/absence survey. METHODS: A presence/absence non-random survey was conducted in which pooled faeces from lambs from 50 farms in the lower and central North Island (n=22) and lower South Island (n=28) were cultured, with and without a post-culture period of chilling, to produce third-stage parasitic nematode larvae (L3). After recovery using the Baermann technique, individual L3 were identified to Nematodirus spp. using a PCR assay. This identified the species that were present on each farm that were then used to compare the regions. RESULTS: Nematodirus filicollis was present in 38/50 (76%) samples from which Nematodirus spp. larvae were cultured, compared with 50/50 (100%) in the case of N. spathiger. No regional differences were observed in the prevalence of either species (p=0.74). Of the Nematodirus spp. L3 recovered from the unchilled samples, 415/428 (97%) were N. spathiger and 13/428 (3%) N. filicollis. After chilling 594/695 (85%) of the Nematodirus spp. L3 were N. spathiger and 101/695 (15%) were N. filicollis. CONCLUSION: Despite the non-random nature of the study, the evidence suggests that both these species are likely to occur sympatrically on most sheep farms throughout New Zealand. In general N. filicollis eggs did not hatch without a period of chilling and this has implications for the identification of these larvae using conventional culture methods.

Acquired immunity to endoparasites in sheep interacts with anthelmintic treatment to influence selection for anthelmintic resistance.

Abstract AIM: To test the hypothesis that anthelmintic treatment of animals with a well-developed immunity to endoparasites will be more selective for anthelmintic resistance than treatment of animals that are not immunocompetent. METHODS: In Experiment 1, five pregnant, mixed-aged ewes, and five newly weaned 3-month-old lambs were housed indoors, and infected three times a week with a mixture of albendazoleresistant and -susceptible Teladorsagia (=Ostertagia) circumcincta larvae for 12 weeks. In Experiment 2, six two-tooth ewes, six 9-month-old lambs, and 10 newly weaned 3-month-old lambs were infected three times a week with a mixture of albendazoleresistant and -susceptible Trichostrongylus colubriformis larvae for 10 weeks over the winter months of June to August. Egghatch assays (EHA) were performed on a weekly basis, to determine the resistance status of eggs passed before and after a treatment with albendazole, that was given at least one week after the infections became patent. RESULTS: Faecal nematode egg counts (FEC) indicated that mature ewes and immunocompetent lambs restricted the establishment of parasite larvae, especially after the anthelmintic treatment. Although EHA were completed for T. circumcincta, the numbers of eggs recovered from the ewes were too few to allow accurate comparisons of anthelmintic resistance status between ewes and lambs. In Experiment 2, viable infections of T. colubriformis only developed in five 9-month-old and five 3-month-old lambs after anthelmintic treatment. Comparison of the concentrations of anthelmintic needed to kill 50% of the eggs (LC50) from the lambs showed that where host immunity restricted the establishment of new infection after anthelmintic treatment, the resistance status of eggs passed remained high until the end of the study. In contrast, where new infections established, the resistance status of eggs in the faeces declined to levels close to those prior to anthelmintic treatment. CONCLUSIONS: The results support the conclusion that older animals are more refractory to the establishment of ingested parasite larvae, and that this reduces the dilution of resistant worms surviving an anthelmintic treatment. Surviving resistant worms are therefore likely to make a greater proportional contribution to the resulting population of parasites on pasture. The findings support the view that treating mature sheep with an anthelmintic should be considered a higher-risk practice for selecting anthelmintic-resistant parasites, than treating lambs.

Benzimidazole resistance in Nematodirus spathiger and N. filicollis in New Zealand

Abstract AIM: To determine the prevalence of benzimidazole resistance in Nematodirus spathiger and N. filicollis from a sample of New Zealand farms. METHODS: The efficacy of albendazole (ABZ) against Nematodirus spp. was assessed by faecal nematode egg count reduction (FECR) tests undertaken in lambs aged 3–8 months old on 27 sheep farms throughout New Zealand. On each farm, groups of 10–16 lambs were either treated with ABZ (4.75 mg/kg) or remained as untreated controls. Faecal samples were collected from all animals at the time of treatment and 7–10 days later. Faecal nematode egg counts (FEC) were performed using a modified McMaster technique. Larvae were cultured from pooled faecal samples, collected 7–10 days after treatment from each group, by incubation at 20°C for 6 weeks, 4°C for 26 weeks then 13°C for 2 weeks. The resulting third stage larvae were identified to species using a multiplex PCR assay, that identified species-specific sequences in the second internal transcribed spacer region of ribosomal DNA. The efficacy of ABZ for N. spathiger and N. filicollis was calculated from the proportion of the two species in culture and the group mean FEC before and after treatment. Only farms with a mean of 10 epg for each species in untreated samples were included for analysis. Resistance was defined as an efficacy <95%. RESULTS: On farms that met the threshold of 10 epg in faecal samples, benzimidazole resistance was found on 20/21 (95%) farms for N. spathiger compared with 4/10 (40%) farms for N. filicollis (p<0.05). In samples collected following treatment, a mean of 83 (min 46, max 100)% of Nematodirus spp. larvae recovered from the untreated groups were N. spathiger, compared with 94 (min 45, max 100)% in the ABZ treated groups (p=0.03). This change in percentage was not influenced by the overall efficacy of treatment based on the FECR test (p=0.324). CONCLUSION: The results confirm the high level of resistance in N. spathiger in New Zealand and that benzimidazole resistance was more common in N. spathiger than N. filicollis. While resistance to benzimidazole anthelmintics has been reported previously in New Zealand, this is the first report of N. filicollis being resistant to benzimidazole anthelmintics.

Carbendazim, at concentrations used on pasture for facial eczema control, reduces development of Trichostrongylus colubriformis when sprayed onto infected sheep faeces.

Abstract AIM: To determine whether the fungicide, carbendazim, as applied to pastures for controlling facial eczema (FE), would inhibit development of the free-living stages of the gastrointestinal nematode parasite Trichostrongylus colubriformis. METHODS: Two studies were conducted, using sheep faeces containing eggs of T. colubriformis. In the first, the faeces were either exposed or not to an application of carbendazim sprayed at the recommended rate for FE control. After spraying, dishes containing the faeces were incubated at 20°C for 14 days, and the resulting third-stage infective larvae (L3) extracted by baermannisation and counted. In addition, naturally infested pasture was also sprayed, and the number of L3 present 7 days later was assessed by cutting herbage samples and extracting larvae by soaking in water and baermannisation. In the second, the faeces were incubated at 20°C for 0, 3 or 7 days before being exposed to no, one or two applications of carbendazim. After further incubation for 14, 11 or 7 days, L3 were similarly extracted by baermannisation and counted. RESULTS: In the first study, there was a 74% reduction in the number of T. colubriformis larvae recovered from faeces exposed to carbendazim compared with faeces not exposed, but there was no reduction in the number of L3 recovered from herbage. In the second study, faeces incubated for 0 or 3 days prior to exposure to a single application of carbendazim yielded 98% or 89% fewer larvae, respectively, than faeces not exposed. Faeces incubated for 7 days prior to exposure yielded similar numbers of larvae to faeces not exposed. CONCLUSION: Treatment of pastures with carbendazim for FE control is likely to result in reduced development of the larvae of T. colubriformis, and by inference those of other species, where the application coincides with the presence of freshly deposited faeces containing eggs and developing larvae. However, no effect of treatment on L3 was indicated. The significance of this for on-farm nematode parasite control remains to be determined, as does any potential for strategic applications of carbendazim to pasture aimed at reducing numbers of parasite larvae on pasture. The latter should not be contemplated without due consideration of the implications for the development of anthelmintic resistance.