Simultaneous resistance to multiple anthelmintic classes in nematode parasites of cattle in New Zealand.

Resistance to the benzimidazole and macrocyclic lactone anthelmintics is widespread in Cooperia spp. on cattle farms in New Zealand. Since this was first documented in 2006 little has changed in cattle farming systems except for the widespread use of levamisole to control Cooperia spp. in young cattle (i.e., parasite control has maintained an almost total reliance on use of anthelmintics). Here we report the emergence of simultaneous resistance to the benzimidazole, macrocyclic lactone and levamisole anthelmintics in Cooperia spp. and in Ostertagia spp. Anthelmintic efficacy against nematode parasites of cattle was investigated on four commercial farms following reports of poor animal growth rates and welfare, and positive faecal egg counts, despite routine treatment with combination anthelmintics, which included levamisole. Faecal egg count reduction tests involved 15 animals per treatment group, individual egg counts (paired samples) conducted pre- and post-treatment, with eggs counted to ≤ 15 eggs per g faeces and larval cultures for morphological identification. Actives tested varied between farms but always included levamisole alone and several combination products containing levamisole. Of the 20 tests conducted (i.e., 5 products on each of 4 farms) only 3 exceeded 90% efficacy against Cooperia spp. even though 8 of the products tested were combinations containing levamisole and at least one other broad-spectrum anthelmintic. Levamisole used alone achieved efficacies between 44% and 71% against Cooperia spp. across the four trials. The only product to exceed 95% efficacy against Cooperia spp. was a combination of monepantel + abamectin which was 100% effective against all parasites. Resistance to oxfendazole in Ostertagia spp. was indicated on 3 farms, while on one farm efficacy of all the tested products was ≤75% against this parasite. All the farms involved in this study were farming intensive cattle operations with an almost total reliance on anthelmintics to control parasitism. The results clearly demonstrate the emergence of simultaneous resistance to oxfendazole, levamisole and the macrocyclic lactone anthelmintics. Despite years of advice and recommendations to change farming practices away from intensive monocultures, many farmers have continued with the practice, and some are now faced with the very real possibility of being unable to control cattle parasites on their farms.

Worms and welfare: Behavioural and physiological changes associated with gastrointestinal nematode parasitism in lambs.

Parasitism with gastrointestinal nematodes (GIN) is a worldwide issue impacting negatively on animal production, health, and welfare. Therefore, early diagnostic signs of parasitism are required to allow for timely interventions. The objective of this study was to evaluate the behavioural and physiological changes in lambs associated with GIN infection. We used 30, 8-month-old Romney-cross wethers, that were administered anthelmintics until faecal egg counts (FEC) were zero and housed in an indoor facility. The study lasted 9 weeks, which comprised a 3-week pre-treatment, and a 6-week treatment phase. Lambs were randomly assigned to one of two treatments (n = 15/treatment) trickle-dosed with: 1) 1500 infective third stage larvae (L3) three days/week for 6 weeks (27,000 total L3; challenged), or 2) water 3 days/week for 6 weeks (control). Within each pen there were 5 pairs of lambs (balanced for liveweight), with each pair comprising a challenged and control lamb. Blood, faecal, and saliva samples were collected 1 week pre-treatment and weekly for 6 weeks of treatment. Behaviour was observed (e.g., feeding, lying, standing) from video-camera recordings using scan sampling every 5 min for 8 h, 1 day pre-treatment and on the day immediately prior to physiological sampling across the 6-week treatment phase (7 days in total). Accelerometers were attached to each lamb to continuously monitor behaviour from 3 weeks pre-treatment and for the remainder of the study. Liveweight, body condition, faecal soiling and faecal consistency scoring were performed weekly as was lipidomic analysis of plasma samples. From week 2 of treatment, challenged lambs spent less time feeding and more time lying than control lambs until week 5 of treatment (P ≤ 0.01). At week 3 of treatment, elevated lipids (mainly triglycerides and phospholipids), loose faeces and faecal soiling around the anus were observed in challenged lambs compared with controls (P ≤ 0.05). From week 4 of treatment, FEC were elevated in the challenged compared to control lambs (P ≤ 0.05). There was also lower liveweight gain at 4 and 5 weeks of treatment in the challenged lambs compared with control lambs (P ≤ 0.05). These results show a clear timeline of changes in behaviour (e.g., feeding and lying), lipids such as triglycerides, and digestive function (e.g., faecal soiling) suggestive of GIN subclinical disease, which show promise for use in future studies on early identification of subclinical GIN parasitism in lambs.

In vitro evaluation of fitness parameters for isolates of Teladorsagia circumcincta resistant and susceptible to multiple anthelmintic classes.

Anthelmintic resistance (AR) is an ever increasing problem for the sheep industry. Several studies worldwide have investigated reversing the trend of increasing AR and documented evidence for reversion toward susceptibility has been found. The hypothesis that resistance mutations compromise parasite fitness was drawn from this evidence. The aim of this study was to assess whether there were measurable differences in the fitness of Teladorsagia circumcincta isolates depending on their AR status. Four isolates were selected for the trial based on their known resistance status; D and M were multi-drug resistant, and T and W were susceptible to the benzimidazole, levamisole, and macrocyclic lactone anthelmintic classes. A secondary aim was to develop a series of in vitro bioassays for assessing fitness characteristics of parasites. The in vitro assays included; the cold stress test measured the number of third stage larvae (L3) developing from eggs stored at 4 °C for different lengths of time. Larval aging measured the locomotory activity of L3 after storage at 30 °C for different lengths of time. The exsheathment assay measured the exsheathment percentage of L3. Larval Length used length as a proxy for fecundity. The egg hatch assay evaluated egg hatch rate in water at room temperature. All isolates exhibited a decrease in the number of L3 recovered after storage of eggs at 4 °C (p < 0.001). Storage of L3 at 30 °C significantly influenced the ability of L3 to migrate through a 20 µm sieve (p < 0.001), however, there were no differences between isolates (p > 0.05). Exsheathment rate was higher for isolate D in comparison to isolates M and W, and for isolate T compared to isolate W. Isolate W was significantly longer than all other isolates (p < 0.05), whilst isolate M was significantly longer than isolate D (p < 0.05). No significant differences were found between isolates in egg hatch (p > 0.05). Overall, the results do not support differences in fitness associated with anthelmintic resistance status, even though differences were seen between the isolates for some assays. This suggests there is considerable variation in fitness parameters between isolates, making it difficult to determine whether resistance genotypes come with lower fitness.

Host effects on the free-living stages of Haemonchus contortus.

A group of 5 lambs (Host 1-5) was infected with the same batch of Haemonchus contortus and after patency individual faecal samples were collected, separately incubated at 23 °C for 14 days and third stage larvae collected through Baermannisation. Life-history traits were compared between larvae from different hosts: the length of the larvae was measured by microscope image analysis, larval survival in water at 35 °C, larval susceptibility to ivermectin (EC50) in a migration assay, the proportion of larvae exsheathing in vitro and the proportion establishing to the adult stage in young lambs. For all traits there were significant differences between the host animals, with larvae from specific hosts following a consistent pattern of displaying the highest or lowest trait results. Compared with larvae from Host 1 the larvae from Host 5 were () shorter (741-692 µm, p < 0.05), had a longer median survival at 35 °C (3.6-6.4 days, p < 0.05), were less susceptible to ivermectin (EC50 of 1.2 v 4.5 µM, p < 0.05), exsheathed to a lesser degree (83.6-58 %, p < 0.05), but showed a higher establishment rate in the consecutive host (15.2-31.4 %, p < 0.05). Regarding the survival time, anthelmintic susceptibility (under most commercial farming practices) and establishment rate as indicators for fitness, the parasites populating Host 5 produced progeny of higher fitness. The findings indicate that the host animal of the parental parasite generation has a significant effect on the parasite progeny.

Monitoring equine ascarid and cyathostomin parasites: Evaluating health parameters under different treatment regimens.

BACKGROUND: Strongylid and ascarid parasites are omnipresent in equine stud farms, and ever-increasing levels of anthelmintic resistance are challenging the industry with finding more sustainable and yet effective parasite control programs. OBJECTIVES: To evaluate egg count levels, bodyweight and equine health under defined parasite control protocols in foals and mares at two Standardbred and two Thoroughbred stud farms. STUDY DESIGN: Longitudinal randomised field trial. METHODS: A total of 93 foals were enrolled and split into two treatment groups, and 99 mares were enrolled and assigned to three treatment groups. All horses underwent a health examination, and episodes of colic or diarrhoea were recorded at each faecal collection date. Bodyweights were assessed using a weight tape, and mares were body condition scored. Group A foals (FA) were dewormed at 2 and 5 months of age with a fenbendazole/ivermectin/praziquantel product, while group B foals (FB) were dewormed on a monthly basis, alternating between the above-mentioned product and an oxfendazole/pyrantel embonate product. Group A mares (MA) were dewormed twice with fenbendazole/ivermectin/praziquantel, group B mares (MB) were dewormed with the same product, when egg counts exceeded 300 strongylid eggs per gram, and group C mares (MC) were dewormed every 2 months, alternating between the two products. Health data were collected monthly for 6 months (foals) and bimonthly for 13 months (mares). Data were analysed with mixed linear models and interpreted at the α = 0.05 significance level. RESULTS: There were no significant bodyweight differences between foal groups, but MA mares were significantly lighter than the other two groups. Very few health incidents were recorded. Foals in group FA had significantly higher ascarid and strongylid egg counts, whereas no significant differences were observed between mare groups. MAIN LIMITATIONS: Study duration limited to one season. CONCLUSIONS: Anthelmintic treatment intensity was lowered from the traditional intensive regimes without measurable negative health consequences for mares and foals.

Abomasal nematode species differ in their in vitro response to exsheathment triggers.

A crucial step in the infection process of grazing ruminants by gastro-intestinal nematodes is the exsheathment of the infective third-stage larva following ingestion. Recently, heat shock was shown to play an important role in the carbon dioxide (CO2)-dependent exsheathment response in Haemonchus contortus. The current in vitro study set out to evaluate the role of heat shock in other abomasal species. In rumen fluid, all species tested exsheathed rapidly and efficiently in response to heat shock and CO2. This response was significantly higher compared to slow temperature changes, supporting the hypothesis that heat shock plays an important role in vivo. However, in artificial buffer, the effect of heat shock was species-dependent. For H. contortus and Ostertagia leptospicularis, the response in artificial buffer was similar to rumen fluid. In contrast, Ostertagia ostertagi and Teladorsagia circumcincta exsheathment was significantly lower and/or slower in artificial buffer, and there was no benefit of heat shock. For these two species, it appears that there are co-factors in the rumen fluid, in addition to heat shock and CO2, contributing to exsheathment. Overall, the data indicate that there are significant differences between abomasal species in their response to exsheathment triggers.

Carbon dioxide is an absolute requirement for exsheathment of some, but not all, abomasal nematode species.

The first step in the infection process of grazing ruminants by gastrointestinal nematodes is the exsheathment of the third-stage larva (L3). Exsheathment of various species can be achieved in vitro using carbon dioxide (CO2) under the appropriate temperature and pH conditions. However, it remains unclear whether elevated CO2 levels are an absolute requirement for exsheathment. Exsheathment of four abomasal species was investigated in both the presence and absence of CO2, in either rumen fluid (cow or sheep) or buffer (standard or enriched). Exsheathment of Ostertagia ostertagi, Teladorsagia circumcincta and Ostertagia leptospicularis was observed in CO2-depleted rumen fluid and enriched buffer (respectively 46%, 22% and 15% in rumen fluid and 28% 18% and 26% in enriched buffer after 24 h). The level of this response was dependent on the species as well as the medium, and exsheathment was significantly higher in the presence of CO2. For Haemonchus contortus, exsheathment could only be achieved under CO2-saturated conditions. In conclusion, even though these parasite species exsheath in the same environment, there were significant differences in the minimal requirements to trigger their exsheathment. Some abomasal species were capable of exsheathment in the absence of CO2, which is likely facilitated by cofactors present in the rumen fluid and/or enriched buffer.

Heat shock, but not temperature, is a biological trigger for the exsheathment of third-stage larvae of Haemonchus contortus.

Gastrointestinal parasites are an important health issue in grazing ruminants. Understanding the processes involved in the transition from the free living to the parasitic life stage of these nematodes is one avenue to identifying new targets amenable to future intervention. The transition to parasitism is initiated by exsheathment and is triggered by the sudden change in environment after ingestion of the infective larva by the host. Two major changes in environment are the increases in temperature and carbon dioxide (CO2) levels. For CO2 a role in exsheathment has been described previously, but the exact role of temperature was unclear. The current study is the first to investigate the importance of temperature in triggering exsheathment of Haemonchus contortus. Carbon dioxide induced exsheathment in H. contortus proved to be temperature dependent, as no exsheathment was observed at room temperatures. However, the temperature requirement to trigger exsheathment was quite specific. A rapid change in temperature (heat shock) very efficiently induced high levels of exsheathment. In contrast, when the larvae were exposed to a slow increase in temperature, the exsheathment response was smaller and delayed. Further investigation revealed that timing of the heat shock in relation to the CO2 administration was crucial, as well as the final temperature and magnitude of the heat shock. In conclusion, these data indicate that heat shock rather than temperature itself is a crucial aspect in triggering the biological exsheathment cascade, and thus infection process, of H. contortus.

The effect on liveweight gain of using anthelmintics with incomplete efficacy against resistant Cooperia oncophora in cattle.

A replicated field trial was conducted to measure the effect on liveweight gain of failing to adequately control anthelmintic resistant populations of Cooperia oncophora and to determine whether populations, and hence production losses, increased with time. Eight mobs of 10 Friesian-Hereford calves were run on independent farmlets from January to December, over each of two years. All mobs were routinely treated with a pour-on formulation of eprinomectin every six weeks, which controlled parasites other than Cooperia. Four mobs also received six weekly treatments with an oral levamisole plus albendazole combination anthelmintic to control Cooperia. Liveweights, condition scores, faecal egg counts and larval numbers on pasture were measured throughout. In the first year animals treated with eprinomectin alone were 12.9 kg lighter in November than those treated with eprinomectin plus albendazole and levamisole, however, in the second year there was no difference between the treatment groups. The data, therefore, support the view that while C. oncophora is less pathogenic than other cattle parasite species it can still cause production losses when present in sufficient numbers. In the first year of the study, parasite load, as measured by faecal nematode egg count and larval numbers on herbage, tended to be higher and calf growth rates lower than in the second year. In both years, counts of infective larvae on herbage declined over winter-spring to be at low levels before mid-summer. This suggests that the carry-over of infection from one crop of calves to the next was relatively small and hence that the level of challenge to the young calves at the start of each year was largely due to the effectiveness of the quarantine treatments administered when the animals arrived on the trial site. Low survival of larvae on pasture between grazing seasons, resulting in small larval populations on pasture when drenching programmes start each summer, might help to explain the widespread development of anthelmintic resistance in this parasite under New Zealand grazing systems.

Confirmation of ivermectin resistance in Ostertagia ostertagi in cattle in New Zealand.

Six suspected cases of ivermectin resistance in Ostertagia spp. in cattle were investigated after routine anthelmintic efficacy testing on commercial farms. On four farms a comprehensive faecal egg count reduction test (FECRT) was undertaken using oral formulations of ivermectin (0.2mg/kg), albendazole (10mg/kg) and levamisole (7.5mg/kg) while on two farms only ivermectin was tested. The proportions of Ostertagia spp. in the untreated control and post-treatment larval cultures were used to apportion egg counts to genera and determine efficacy against this genus. Isolates of Ostertagia spp. recovered from three of the farms were each used to infect 18 six month old calves. The efficacy of oral formulations of ivermectin and moxidectin, both at 0.2mg/kg, was determined against each isolate by slaughter and worm count. The efficacy of ivermectin against Ostertagia spp., based on differentiated FECRT for each of the farms varied from 0% to 88%. The efficacy of ivermectin based on worm counts in the slaughter trial varied from 13% to 75% but moxidectin was >99% effective against all isolates. In addition, in the FECRT albendazole, at a dose rate of 10mg/kg, failed to achieve 95% efficacy against Ostertagia spp. on two farms (82% and 85%). Levamisole consistently failed to achieve 95% efficacy against Ostertagia spp. which is consistent with its known lesser efficacy against this parasite. These results confirm the presence of macrocyclic lactone resistant O. ostertagi in cattle in New Zealand and the likely presence of dual resistance, to macrocyclic lactones and albendazole, in some isolates. Resistant populations of this highly pathogenic parasite are probably not uncommon in New Zealand and pose a significant threat to animal production and welfare in the future.

Evidence for reversion towards anthelmintic susceptibility in Teladorsagia circumcincta in response to resistance management programmes.

Maintaining production and economic viability in the face of resistance to multiple anthelmintic actives is a challenge for farmers in many countries. In this situation, most farmers in New Zealand rely on the use of combination products, containing multiple actives with similar spectra of activity, in order to maintain control. However, there are concerns that use of combinations, once resistance has already developed to the individual actives, could rapidly lead to complete failure of all actives. This study followed seven farms, previously diagnosed with resistance to at least two classes of anthelmintic, which were implementing a tailored programme of 'best practice parasite management'. The aim was to ascertain whether the programmes, which included the almost exclusive use of combination anthelmintics, were able to prevent resistance from developing further. Strategies implemented on each farm varied, but had consistent underlying principles i.e. to avoid over-use of anthelmintics; to minimise parasite challenge to susceptible stock; to maintain refugia of susceptibility and to ensure that only effective anthelmintics were used. Annual faecal egg count reduction tests (FECRT) were undertaken in lambs on all farms to monitor anthelmintic efficacy over 5 years. The efficacy of albendazole, ivermectin and levamisole was calculated and the changes in efficacy against Teladorsagia circumcincta assessed. Overall, there was a significant improvement in the effectiveness of both levamisole and ivermectin against T. circumcincta, and a positive but non-significant trend in efficacy of albendazole, i.e. there was evidence for reversion towards susceptibility. Hence, the almost exclusive use of combination anthelmintics, integrated with other resistance management strategies, did not result in further resistance development despite all farms exhibiting resistance to multiple actives at the outset. What-is-more, the measured increases in anthelmintic efficacy suggests that adoption of best practice management strategies may extend the useful life of anthelmintics even after resistance has been diagnosed.

Influence of nematophagous fungi, earthworms and dung burial on development of the free-living stages of Ostertagia (Teladorsagia) circumcincta in New Zealand.

Biological options for nematode parasite control are being sought, as the long-term efficacy of conventional anthelmintics comes increasingly under threat from drug-resistant parasites. Three biological methods with the potential to reduce pasture contamination by parasitic nematode larvae were examined: (a) killing of larvae developing in dung by nematophagous fungi; (b) removal of dung through earthworm ingestion; (c) burial of dung in soil as might occur through the action of dung beetles. Field trials with the test bio-control agents were carried out in autumn and spring by adding dung from sheep infected with Ostertagia (Teladorsagia) circumcincta to pots of ryegrass/white clover. The factorial treatment structure included five fungal treatments (individual applications of Duddingtonia flagrans, Monacrosporium gephyropagum and Harposporium helicoides, a combination of all the three fungi together and an untreated control), two dung burial treatments (dung buried or deposited on the soil surface) and two earthworm treatments (earthworms present or absent). D. flagrans and H. helicoides, individually or in combination, reduced recovery of infective stage larvae in experiment 1, while only H. helicoides reduced recovery in experiment 2. In both the experiments, dung burial increased the total number of larvae recovered, while the number of infective larvae were reduced by the action of earthworms. Increased recovery following burial, along with the fact that larvae moved rapidly from soil onto herbage, suggests that soil may provide a protective reservoir for infective larvae infesting herbage.