Chilling requirements for hatching of a New Zealand isolate of Nematodirus filicollis.

The eggs of some species of the parasitic nematode Nematodirus require a period of chilling before they can hatch; N. filicollis is one such species. This study investigated this requirement for chilling in a New Zealand strain of this species. Eggs of N. filicollis were extracted from lamb's faeces and incubated at 20°C to allow development to the third stage larvae within the egg. These eggs were then placed into tissue culture plates and incubated at: 2.7°C (±0.99), 3.6°C (±0.90), 4.7°C (±0.35), 6.4°C (±0.37), 8.0°C (±1.54) or 9.9°C (±0.14) for up to 224 days. At 14day intervals until day 84, then every 28 days, one plate was removed from each temperature and placed at 13.1°C (±0.44) for 14 days. Eggs were then assessed for hatching. From this data, chill units were calculated by subtracting the culture temperature from a constant threshold of 11°C and multiplying by the number of days for which the sample was cultured; then the Gompertz model fitted. Even though hatching overall was low, a greater proportion of eggs hatched with chill accumulation. Maximum hatching of eggs required 800-1000 chill units. Consequently in the field, more than one season of chilling would be required before hatching. As such a generation time could take more than one year to complete. This is different to the hatching dynamics of N. spathiger, the other main species found in New Zealand sheep, which does not display this requirement for chilling and hatches immediately once the third stage larvae are developed.

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

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.

Managing anthelmintic resistance--use of a combination anthelmintic and leaving some lambs untreated to slow the development of resistance to ivermectin.

A field study was conducted to test the hypotheses that use of a combination anthelmintic and/or increasing the pool of unselected worms 'in refugia' by leaving a proportion of lambs untreated would slow the development of resistance to ivermectin. Twelve suites of four paddocks (farmlets) were seeded with a mixture of resistant and susceptible isolates of both Trichostrongylus colubriformis and Teladorsagia (Ostertagia) circumcincta calculated to yield a 95% reduction in faecal nematode egg count (FEC) after treatment with ivermectin. Each year for three years the farmlets were stocked in the spring with mobs of lambs which were treated five times at 28-day intervals with either ivermectin or an ivermectin+levamisole combination. In addition, in half the mobs the heaviest 10% of lambs remained untreated at each treatment occasion, resulting in a 2 × 2 factorial treatment structure (i.e. two drench types × two percentage treated) with three complete replicates. The development of resistance to ivermectin, and at the end to levamisole, was measured by larval development assays (LDA) and worm counts from treated and untreated tracer lambs. For T. colubriformis the development of resistance to ivermectin, as measured by tracer lamb worm burdens, was delayed by treatment with the combination and by leaving 10% of lambs untreated. In addition, the interaction between these factors approached significance (p=0.052). Similarly, results of the LDAs indicated a slower development of resistance when lambs were treated with the combination and when 10% of lambs were left untreated. For T. circumcincta, results were compromised by the rapid development of resistance, which appears to be the result of low viability in the field of the susceptible isolate used to contaminate the pastures. Although a small delay in the development of resistance to ivermectin was indicated, this was off-set by an increase in the level of resistance to levamisole. A post-study modelling experiment simulating the conditions of the field study and the starting efficacies for the two nematode species produced equivalent outputs to those measured in the field. Overall, results support the conclusions that use of combination anthelmintics and deliberately increasing 'refugia' of unselected genotypes will slow the development of anthelmintic resistance. However, as indicated in modelling studies, once resistance to all the constituent actives is well developed (efficacy<70%) the value of combinations for slowing the development of resistance is largely lost.

The production cost of anthelmintic resistance in lambs.

The economic impact of anthelmintic resistance was investigated in lambs by comparing productivity parameters in groups of animals treated either with a highly effective anthelmintic, or an anthelmintic to which three species of resistant worms were known to be present. Ten farmlets, each stocked with 30 lambs, were rotationally grazed for 5 months, with monthly treatments of either albendazole, to which resistance existed, or a new combination product containing derquantel and abamectin (DQL-ABA) to which there was no resistance. Stock on five farmlets were treated with each anthelmintic and productivity measures, including liveweights, body condition and faecal soiling were assessed throughout. In addition, fleece weights and information on carcass weight and quality was collected at the end of the trial. Anthelmintic efficacy was measured at the last two treatment dates by faecal egg count reduction test with larval cultures. Albendazole demonstrated efficacies of 48.4% and 40.9% for Trichostrongylus spp. and Teladorsagia circumcincta respectively. By contrast, the DQL-ABA treatments were >99% effective against all genera. The difference in live-weight gain was 9 kg in favour of the DQL-ABA treatments. This translated into a 4.7 kg increase in carcass weight with a 10.4% increase in carcass value. Significant differences in body condition scores, faecal breech soiling and fleece weights were also recorded, all in favour of the DQL-ABA treatments. The time required for 50% of the animals to reach a target live-weight of 38 kg was significantly shorter (by 17 days) in those animals treated with DQL-ABA. The results show that the production cost of subclinical parasitism as a result of using an anthelmintic product which is less than fully effective due to resistance can greatly exceed the cost of routine testing of anthelmintic efficacy and the adoption of new anthelmintic classes. There is a strong case for many farmers to re-evaluate their position on some of these issues in order to optimise financial performance.

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

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 of L3 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 to have 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.

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.

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 best predictor 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, over the 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 of New 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 contortus appeared 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.

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

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 albendazole resistant 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 albendazole resistant and -susceptible Trichostrongylus colubriformis larvae for 10 weeks over the winter months of June to August. Egg-hatch 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 (LC(50)) 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.

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

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 manufacturer's 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 egg-hatch 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.

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

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 degrees 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 degrees 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.