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.

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.