Supplementation of minerals and vitamins influences optimal targeted selective treatment thresholds for the control of gastro-intestinal nematodes in lambs.

Targeted Selective Treatment (TST) is a gastrointestinal nematode (GIN) control strategy where anthelmintic treatment decisions are made at an individual animal level. TST has been proven to reduce anthelmintic use and subsequently slow down anthelmintic resistance development, however questions remain regarding optimal TST methods and their applicability across farms. In this study, the influence of Mineral and Vitamin (MV) supplementation on optimal energy utilisation (EU) TST thresholds was assessed on three Welsh farms. In total, 360 lambs were split into two groups, MV supplemented and control, and were treated with an anthelmintic against GIN at the midway point of the experiment. Lambs that improved their EU efficiency post treatment were deemed to have benefited from anthelmintic treatment. Optimal EU TST thresholds was determined for each treatment group per farm using Youden's J statistic where the treatment threshold retrospectively exhibiting the greatest combined sensitivity and specificity in correctly identifying lambs benefiting from treatment was deemed to be optimal. Results demonstrated that the optimal EU TST threshold was higher in MV supplemented groups at 0.72, 0.71 and 0.56 versus 0.58, 0.67, 0.51 for control groups on each respective farm. Identification of lambs for TST was more effective when using an optimised EU TST threshold, compared to when using the standard EU TST threshold of 0.66. The study highlights that applying standard EU TST thresholds may not be appropriate on all commercial farms with factors including MV status as noted in this study likely to influence optimal EU TST thresholds. Additional refinement of TST systems can further strengthen their applicability across sheep flocks.

Associations between Gastrointestinal Nematode Infection Burden and Lying Behaviour as Measured by Accelerometers in Periparturient Ewes.

The application of precision livestock farming (PLF) technologies will underpin new strategies to support the control of livestock disease. However, PLF technology is underexploited within the sheep industry compared to other livestock sectors, and research is essential to identify opportunities for PLF applications. These opportunities include the control of endemic sheep disease such as parasitic gastroenteritis, caused by gastrointestinal nematode infections, which is estimated to cost the European sheep industry EUR 120 million annually. In this study, tri-axial accelerometers recorded the behaviour of 54 periparturient Welsh Mule ewes to discover if gastrointestinal nematode (GIN) infection burden, as measured by faecal egg count (FEC), was associated with behavioural variation. Linear mixed models identified that increasing FECs in periparturient ewes were significantly associated with a greater number of lying bouts per day and lower bout durations (p = 0.013 and p = 0.010, respectively). The results demonstrate that FECs of housed periparturient ewes are associated with detectable variations in ewe behaviour, and as such, with further investigation there is potential to develop future targeted selective treatment protocols against GIN in sheep based on behaviour as measured by PLF technologies.

Exploration of factors associated with spatial-temporal veterinary surveillance diagnoses of rumen fluke (Calicophoron daubneyi) infections in ruminants using zero-inflated mixed modelling.

Rumen fluke (Calicophoron daubneyi) has emerged as a prominent parasite of ruminants in Europe over the past decades. Epidemiological questions remain regarding this observed increase in prevalence as well as the prospect for future paramphistomosis risk. This study aimed to identify factors associated with the temporal−spatial prevalence of rumen fluke as measured by veterinary surveillance in a temperate region using zero-inflated negative binomial mixed modelling. Modelling revealed that summer rainfall, raindays and sunshine hours and mean winter temperature as significant positively associated climate variables for rumen fluke prevalence over space and time (P < 0.05). Rumen fluke prevalence was also higher in counties with higher cattle/sheep densities and was positively associated with rumen fluke case rates in the previous years (P < 0.05). Equivalent models for fasciolosis prevalence revealed no significant association with winter temperature and sunshine hours, (P > 0.05). These results confirm a strong association between rainfall and the prevalence of both fluke species in a temperate environment, likely due to the role of Galba truncatula as their intermediate snail host. It also highlights the potential added importance of winter temperature and sunshine hours in rumen fluke epidemiology when compared to liver fluke.

Temporal dynamics of trematode intermediate snail host environmental DNA in small water body habitats.

Environmental DNA (eDNA) surveying has potential to become a powerful tool for sustainable parasite control. As trematode parasites require an intermediate snail host that is often aquatic or amphibious to fulfil their lifecycle, water-based eDNA analyses can be used to screen habitats for the presence of snail hosts and identify trematode infection risk areas. The aim of this study was to identify climatic and environmental factors associated with the detection of Galba truncatula eDNA. Fourteen potential G. truncatula habitats on two farms were surveyed over a 9-month period, with eDNA detected using a filter capture, extraction and PCR protocol with data analysed using a generalized estimation equation. The probability of detecting G. truncatula eDNA increased in habitats where snails were visually detected, as temperature increased, and as water pH decreased (P < 0.05). Rainfall was positively associated with eDNA detection in watercourse habitats on farm A, but negatively associated with eDNA detection in watercourse habitats on farm B (P < 0.001), which may be explained by differences in watercourse gradient. This study is the first to identify factors associated with trematode intermediate snail host eDNA detection. These factors should be considered in standardized protocols to evaluate the results of future eDNA surveys.

Gastrointestinal nematode control practices in ewes: Identification of factors associated with application of control methods known to influence anthelmintic resistance development.

Gastrointestinal nematodes (GIN) negatively impact productivity and welfare in sheep globally and are estimated to cost the European sheep industry €157-477 million annually. GIN are mainly controlled by anthelmintic treatment, however, as anthelmintic resistance becomes prominent, the routine treatment of ewes against GIN has been questioned. A questionnaire survey of 383 sheep farmers in Great Britain was conducted to identify strategies currently used to control GIN infections in ewes. Ordinal and binary regression analysis were used to identify factors associated with use of practices known to influence anthelmintic resistance development, including number and timing of ewe GIN anthelmintic treatment, targeted selective treatment (TST) of ewes, drench and move of ewes and long-acting moxidectin treatment of periparturient ewes. Participating farmers treated their ewes against GIN 1.68 times per year on average, with 42.3% and 32.1% of participating farmers worming their ewes once or twice a year on average, respectively. 17.2% of participating farmers wormed their ewes more than twice a year, and 8.4% never worm their ewes. Participating farmers who devised GIN control strategies based on SCOPS guidelines treated their ewes significantly less per year (P < 0.001), whilst those determining treatment timing based on ewe DAG scores or the time of year treated their ewes significantly more frequently (P < 0.001). Farmers who devised GIN control strategies in conjunction with their vet had greater odds of using TST (P < 0.001), as well as farmers who determined flock treatment timing based on ewe condition (P = 0.027). The use of narrow spectrum flukicides was significantly associated with reduced number of annual ewe GIN anthelmintic treatments (P < 0.001), TST of ewes against GIN (P < 0.001) and the avoidance of moving ewes to clean pastures following GIN treatment (P < 0.001). The presence of sheep scab on a farm or in the area was significantly associated with increased annual GIN treatments for ewes (P = 0.002), not using TST strategies to control GIN in ewes (P < 0.001) and moving ewes to clean pasture after anthelmintic treatment, whilst using macrocyclic lactones treatments to prevent sheep scab was significantly associated with the treatment of periparturient ewes with long-acting moxidectin (P = 0.001). This research suggests that by encouraging the application of evidence based targeted or targeted selective treatment strategies, further interaction between farmers and veterinarians/SCOPS guidance, and the uptake of best practices for controlling liver fluke and sheep scab on farms, sustainable GIN control strategies can become the common practice in ewes.

Rapid detection of Galba truncatula in water sources on pasture-land using loop-mediated isothermal amplification for control of trematode infections.

BACKGROUND: Fascioliasis caused by the trematodes Fasciola hepatica and F. gigantica, is a global neglected zoonotic disease estimated to cost the livestock industry over €2.5 billion annually. Farm management measures and sustainable use of anthelmintics can, in principle, effectively control trematode infection in livestock and reduce the rate of developing anthelmintic resistance. Previously, we designed an environmental DNA (eDNA) assay to identify a common trematode intermediate host, the freshwater snail Galba truncatula, in water sources to measure specific trematode infection risk areas on pasture-land. To improve this procedure, we now report a loop-mediated isothermal amplification (LAMP) assay to identify G. truncatula eDNA. METHODS: A LAMP assay was designed and optimised (e.g. temperature, time duration and primer concentration) to identify G. truncatula DNA. The ability of the LAMP assay to target G. truncatula DNA was identified, and LAMP assay limit of detection was investigated in comparison to conventional PCR. In the field, 48 water samples were collected from stream, ditch and water pool habitats in four locations at two Aberystwyth University farms over a seven week period to investigate the applicability of the LAMP assay for use on eDNA samples, in comparison to conventional PCR. RESULTS: The LAMP assay delivered detectable results in 30 min at 63 °C. The assay discriminated between G. truncatula DNA and non-target DNA, presenting a level of DNA detection comparable to conventional PCR. No significant difference was found between the ability of the LAMP and PCR assay to identify G. truncatula eDNA in water samples. Kappa coefficient analysis revealed a moderate level of agreement between LAMP and PCR assays. CONCLUSIONS: This study demonstrated that the LAMP assay can detect G. truncatula eDNA in a simple and rapid manner. The LAMP assay may become a valuable tool to determine optimum pasture management for trematode parasite control.

Detection of Galba truncatula, Fasciola hepatica and Calicophoron daubneyi environmental DNA within water sources on pasture land, a future tool for fluke control?

BACKGROUND: Increasing trematode prevalence and disease occurrence in livestock is a major concern. With the global spread of anthelmintic resistant trematodes, future control strategies must incorporate approaches focusing on avoidance of infection. The reliance of trematodes on intermediate snail hosts to successfully complete their life-cycle means livestock infections are linked to the availability of respective snail populations. By identifying intermediate snail host habitats, infection risk models may be strengthened whilst farmers may confidently apply pasture management strategies to disrupt the trematode life-cycle. However, accurately identifying and mapping these risk areas is challenging. METHODS: In this study, environmental DNA (eDNA) assays were designed to reveal Galba truncatula, Fasciola hepatica and Calicophoron daubneyi presence within water sources on pasture land. eDNA was captured using a filter-based protocol, with DNA extracted using the DNeasy® PowerSoil® kit and amplified via PCR. In total, 19 potential G. truncatula habitats were analysed on four farms grazed by livestock infected with both F. hepatica and C. daubneyi. RESULTS: Galba truncatula eDNA was identified in 10/10 habitats where the snail was detected by eye. Galba truncatula eDNA was also identified in four further habitats where the snail was not physically detected. Fasciola hepatica and C. daubneyi eDNA was also identified in 5/19 and 8/19 habitats, respectively. CONCLUSIONS: This study demonstrated that eDNA assays have the capabilities of detecting G. truncatula, F. hepatica and C. daubneyi DNA in the environment. Further assay development will be required for a field test capable of identifying and quantifying F. hepatica and C. daubneyi infection risk areas, to support future control strategies. An eDNA test would also be a powerful new tool for epidemiological investigations of parasite infections on farms.

The prevalence and development of digenean parasites within their intermediate snail host, Galba truncatula, in a geographic area where the presence of Calicophoron daubneyi has recently been confirmed.

During the past decade, rumen fluke (Calicophoron daubneyi) has established as a prominent parasite of livestock within numerous European countries. Its development and spread is enabled by the presence of its intermediate snail host G. truncatula. However, the dynamics of this stage of the C. daubneyi lifecycle is yet to be recorded in numerous northern European countries including the UK. Here, the prevalence of C. daubneyi along with F. hepatica, H. cylindracea and other parasites infecting G. truncatula snails on 10 Welsh farms was recorded using morphological and PCR techniques. A total of 892 G. truncatula snails were collected between May and October 2016. The prevalence of C. daubneyi in sampled G. truncatula snails (4%) was lower compared to F. hepatica (5.6%). No association in prevalence between these species was recorded. Haplometra cylindracea was found infecting 8.2% of G. truncatula snails, with its prevalence within G. truncatula populations negatively associated with F. hepatica cercariae prevalence (P=0.004). Generalized estimation equation (GEE) linear regression models identified the levels of respective fluke eggs shed onto pasture as the main significant association between prevalence levels of both C. daubneyi and F. hepatica within G. truncatula populations (P<0.001). However, equivalent prevalence levels of C. daubneyi and F. hepatica within G. truncatula populations were associated with higher C. daubneyi egg outputs and lower F. hepatica egg outputs from livestock grazing the G. truncatula habitats. Only one of 36C. daubneyi infected G. truncatula snails was found harbouring its cercarial stages, a significantly lower proportion compared to the 29 of 50 F. hepatica infected G. truncatula harbouring its respective cercariae (P<0.05). These results signify that C. daubneyi may be less adept at infecting and developing in the UK's native G. truncatula populations in comparison with F. hepatica. However, C. daubneyi has previously demonstrated its ability to progressively adapt to an intermediate host in a new environment. If C. daubneyi were to adapt to infect and develop more efficiently in UK G. truncatula populations, paramphistomosis risk would significantly increase leading to increased livestock losses. Questions are also raised regarding potential interaction between digenean species at intermediate snail host level, which could impact subsequent livestock trematodosis risk.

Rumen fluke (Calicophoron daubneyi) on Welsh farms: prevalence, risk factors and observations on co-infection with Fasciola hepatica.

Reports of Calicophoron daubneyi infecting livestock in Europe have increased substantially over the past decade; however, there has not been an estimate of its farm level prevalence and associated risk factors in the UK. Here, the prevalence of C. daubneyi across 100 participating Welsh farms was recorded, with climate, environmental and management factors attained for each farm and used to create logistic regression models explaining its prevalence. Sixty-one per cent of farms studied were positive for C. daubneyi, with herd-level prevalence for cattle (59%) significantly higher compared with flock-level prevalence for sheep (42%, P = 0·029). Co-infection between C. daubneyi and Fasciola hepatica was observed on 46% of farms; however, a significant negative correlation was recorded in the intensity of infection between each parasite within cattle herds (rho = -0·358, P = 0·007). Final models showed sunshine hours, herd size, treatment regularity against F. hepatica, the presence of streams and bog habitats, and Ollerenshaw index values as significant positive predictors for C. daubneyi (P < 0·05). The results raise intriguing questions regarding C. daubneyi epidemiology, potential competition with F. hepatica and the role of climate change in C. daubneyi establishment and its future within the UK.

Confirmation of Galba truncatula as an intermediate host snail for Calicophoron daubneyi in Great Britain, with evidence of alternative snail species hosting Fasciola hepatica.

BACKGROUND: Fasciola hepatica is a highly prevalent parasite infecting livestock in Great Britain, while Calicophoron daubneyi is an emerging parasite within the GB livestock industry. Both F. hepatica and C. daubneyi require an intermediate host snail to complete their life-cycles and infect ruminants; however, there has been no confirmation of the intermediate host of C. daubneyi in GB, while there are questions regarding alternative host snails to Galba truncatula for F. hepatica. In this study, PCR was used to identify C. daubneyi hosting snail species on Welsh pastures and to identify any alternative snail species hosting F. hepatica. FINDINGS: Two hundred and sixty four snails were collected between May-September 2015 from six farms in mid-Wales known to have livestock infected with C. daubneyi and F. hepatica. Fifteen out of 134 G. truncatula were found positive for C. daubneyi, one of which was also positive for F. hepatica. Three snail species were found positive for F. hepatica [18/134 G. truncatula, 13/52 Radix balthica, and 3/78 Potamopyrgus antipodarum (New Zealand mud snail)], but no evidence of C. daubneyi infection in the latter two species was found. CONCLUSION: This study indicates that G. truncatula is a host for C. daubneyi in GB. Galba truncatula is also an established host of F. hepatica, and interactions between both species at intermediate host level could potentially occur. Radix balthica and P. antipodarum were found positive for F. hepatica but not C. daubneyi. This could indicate a role for alternative snail species other than G. truncatula in infecting pastures with F. hepatica in GB.